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Background T. spiralis aspartic protease has been identified in excretion/secretion (ES) proteins, but its roles in larval invasion are unclear. The aim of this study was to characterize T. spiralis aspartic protease-2 (TsASP2) and assess its roles in T. spiralis invasion into intestinal epithelial cells (IECs) using RNAi. Methodology/Principal findings Recombinant TsASP2 (rTsASP2) was expressed and purified. The native TsASP2 of 43 kDa was recognized by anti-rTsASP2 serum in all worm stages except newborn larvae (NBL), and qPCR indicated that TsASP2 transcription was highest at the stage of intestinal infective larvae (IIL). IFA results confirmed that TsASP2 was located in the hindgut, midgut and muscle cells of muscle larvae (ML) and IIL and intrauterine embryos of the female adult worm (AW), but not in NBL. rTsASP2 cleaved several host proteins (human hemoglobin (Hb), mouse Hb, collagen and IgM). The proteolytic activity of rTsASP2 was host-specific, as it hydrolyzed mouse Hb more efficiently than human Hb. The enzymatic activity of rTsASP2 was significantly inhibited by pepstatin A. The expression levels of TsASP2 mRNA and protein were significantly suppressed by RNAi with 5 μM TsASP2-specific siRNA. Native aspartic protease activity in ML crude proteins was reduced to 54.82% after transfection with siRNA. Larval invasion of IECs was promoted by rTsASP2 and inhibited by anti-rTsASP2 serum and siRNA. Furthermore, cell monolayer damage due to larval invasion was obviously alleviated when siRNA-treated larvae were used. The adult worm burden, length of adult worms and female fecundity were clearly reduced in mice challenged using siRNA-treated ML relative to the PBS group, Conclusions rTsASP2 possesses the enzymatic activity of native aspartic protease and facilitates T. spiralis invasion of host IECs., Author summary Trichinellosis has been regarded as a re-emerging or emerging disease, and it is distributed worldwide. Studies investigating T. spiralis ES protein are beneficial to explore potential molecular targets for anti-T. spiralis vaccines. The functions of aspartic protease have been studied in other parasites and demonstrated to be crucial for their parasitism in the host. However, the functions of T. spiralis aspartic protease have not been reported. Here, we expressed and purified a T. spiralis aspartic protease-2 (TsASP2). Our results showed that TsASP2 was expressed in all T. spiralis developmental stages other than NBL and located in the hindgut, midgut and muscle cells of ML and IIL, as well as in areas surrounding embryos within the female uterus. The rTsASP2 possessed aspartic protease activity and functioned to cleave hemoglobin, collagen and IgM. Silencing of the TsASP2 gene could significantly decrease the aspartic protease activity in muscle larva crude proteins, larval invasion of IECs and worm development in the host. We conclude that TsASP2 plays an important role in T. spiralis penetration into host intestinal epithelial cells and could be a candidate vaccine target molecule against T. spiralis infection.

Trypanosome lytic factor (TLF) is a high-density lipoprotein (HDL) subclass providing innate protection to humans against infection by the protozoan parasite Trypanosoma brucei brucei. Two primate-specific plasma proteins, haptoglobin-related protein (Hpr) and apolipoprotein L-1 (ApoL-1), have been proposed to kill T. b. brucei both singularly or when co-assembled into the same HDL. To better understand the mechanism of T. b. brucei killing by TLF, the protein composition of TLF was investigated using a gentle immunoaffinity purification technique that avoids the loss of weakly associated proteins. HDL particles recovered by immunoaffinity absorption, with either anti-Hpr or anti-ApoL-1, were identical in protein composition and specific activity for T. b. brucei killing. Here, we show that TLF-bound Hpr strongly binds Hb and that addition of Hb stimulates TLF killing of T. b. brucei by increasing the affinity of TLF for its receptor, and by inducing Fenton chemistry within the trypanosome lysosome. These findings suggest that TLF in uninfected humans may be inactive against T. b. brucei prior to initiation of infection. We propose that infection of humans by T. b. brucei causes hemolysis that triggers the activation of TLF by the formation of Hpr–Hb complexes, leading to enhanced binding, trypanolytic activity, and clearance of parasites., Author Summary African trypanosomes are parasites that can infect a wide range of mammals, including domestic animals and humans. Several hundred thousand humans are infected with African sleeping sickness, but this number would be much higher if not for a natural defense molecule found in human blood. The trypanosome lytic factor (TLF) is a minor subclass of high-density lipoprotein that contains two proteins found only in primates, apolipoprotein L-1 and haptoglobin-related protein (Hpr). In this paper, we show that Hpr contributes to TLF toxicity to trypanosomes because it binds hemoglobin (Hb). We found that when Hb is bound to TLF, it is rapidly taken up by the parasite and activated within the acidic environment of the parasite's digestive organelle, the lysosome. Within the lysosome, Hb releases iron, inducing a chemical reaction that produces free radicals that damage membranes and contributes to trypanosome killing. Usually, free Hb is rapidly cleared from the circulation of mammals because of the organ damage free Hb can cause. Trypanosome infection results in breakage of red blood cells and the release of large amounts of Hb. We postulate that trypanosome infection causes increased vascular levels of Hb, resulting in the formation of TLF–Hb complexes that may be important in “arming” the human innate immune system to clear the circulation of certain African trypanosomes.

Ticks employ a battery of proteases to digest the contents of host blood meals. Host hemoglobin degradation is facilitated by proteolytic networks in the midgut, the first major region of the body where ingested blood comes into contact with the tick's internal tissues. Our previous studies indicated that HlCPL-A, a cathepsin L-like cysteine protease isolated from the midgut of the ixodid tick Haemaphysalis longicornis, is a potent hemoglobinase, and plays important roles in the digestion of blood acquired from a host. In this paper, we report the effects of silencing of the HlCPL-A gene in H. longicornis using RNA interference (RNAi). We observed that the survival of HlCPL-A-silenced ticks was reduced compared with that of controls during blood digestion, most likely due to the compromised ability of ticks to digest blood. The morphological analysis results of midgut lumen were different between HlCPL-A-silenced ticks and controls, indicating that HlCPL-A plays a crucial role in hemolysis in the midgut of ticks. The expression level was analyzed using quantitative RT-PCR-based endogenous expression approach. Compared to that in malE double stranded RNA (dsRNA)-treated ticks, in the midgut of HlCPL-A dsRNA-treated ticks, some proteases and inhibitors related to the hemoglobin digestive cascade were up-regulated while the others were down-regulated. These results suggest that HlCPL-A is related to the multi-enzyme cascade and protease network for hemoglobin digestion. These findings suggest that the hemoglobin digestive cascade may assemble in the midgut of ticks.

Staphylococcus aureus nasal colonization is an important risk factor for community and nosocomial infection. Despite the importance of S. aureus to human health, molecular mechanisms and host factors influencing nasal colonization are not well understood. To identify host factors contributing to nasal colonization, we collected human nasal secretions and analyzed their ability to promote S. aureus surface colonization. Some individuals produced secretions possessing the ability to significantly promote S. aureus surface colonization. Nasal secretions pretreated with protease no longer promoted S. aureus surface colonization, suggesting the involvement of protein factors. The major protein components of secretions were identified and subsequent analysis revealed that hemoglobin possessed the ability to promote S. aureus surface colonization. Immunoprecipitation of hemoglobin from nasal secretions resulted in reduced S. aureus surface colonization. Furthermore, exogenously added hemoglobin significantly decreased the inoculum necessary for nasal colonization in a rodent model. Finally, we found that hemoglobin prevented expression of the agr quorum sensing system and that aberrant constitutive expression of the agr effector molecule, RNAIII, resulted in reduced nasal colonization of S. aureus. Collectively our results suggest that the presence of hemoglobin in nasal secretions contributes to S. aureus nasal colonization., Author Summary Staphylococcus aureus is a medically important human pathogen that is found in the nasal passages of approximately 1/3 of the population. The nose serves as a reservoir for spread of this pathogen and predisposes the host to potential infection. Factors contributing to S. aureus nasal colonization are only beginning to be elucidated. The collection and analysis of human nasal secretions provided evidence that the presence of hemoglobin in nasal secretions can promote S. aureus nasal colonization. Hemoglobin reduced expression of the S. aureus agr quorum sensing regulatory system known to be involved in surface colonization, and it was found that induction of the agr system reduced nasal colonization. These findings suggest that individuals experiencing frequent nosebleeds would be prone to S. aureus colonization and epidemiological data supports these findings. By understanding host factors and bacterial molecular mechanisms involved in nasal colonization we may one day be able to design novel decolonization strategies.

Background Mutations in the haemoglobin beta-globin (HbB) and glucose-6-phosphate dehydrogenase (G6PD) genes cause widespread human genetic disorders such as sickle cell diseases and G6PD deficiency. In sub-Saharan Africa, a few predominant polymorphic variants of each gene account for a majority of these deficiencies. Examining at a larger scale the clinical importance of these independent genetic disorders, their possible association with malaria pathogenesis and innate resistance, and their relevance for antimalarial drug treatment, would be easier if an accurate screening method with limited costs was available. Methods A simple and rapid technique was developed to detect the most prominent single nucleotide polymorphisms (SNPs) in the HbB and G6PD genes. The method is able to detect the different haemoglobin polymorphisms A, S, C and E, as well as G6PD polymorphisms B, A and A- based on PCR-amplification followed by a hybridization step using sequence-specific oligonucleotide probes (SSOPs) specific for the SNP variants and quantified by ELISA. Results The SSOP-ELISA method was found to be specific, and compared well to the commonly used PCR-RFLP technique. Identical results were obtained in 98% (haemoglobin) and 95% (G6PD) of the tested 90 field samples from a high-transmission area in Tanzania, which were used to validate the new technique. Conclusion The simplicity and accuracy of the new methodology makes it suitable for application in settings where resources are limited. It would serve as a valuable tool for research purposes by monitoring genotype frequencies in relation to disease epidemiology.

Malaria is a widespread endemic disease which contributes significantly to infant mortality. Genetic traits which protect against death by malaria or confer higher fertility on their carriers in malarial environments would have a distinct biologic advantage. As long as malaria remains, such traits could be expected to increase in frequency from generation to generation, so that ultimately the entire population carries the protective gene or genes. The resistance of most West Africans and of their nonimmune descendents in the United States against infections with Plasmodium vivax suggests the presence of such genes in these populations. Their nature and mode of action remains unknown. If a genetic trait protects against malaria but also has harmful effects and causes illness or diminished fertility, an equilibrium gene frequency will be reached at which the beneficial and harmful effects of such a gene balance each other.