Parasite motility is critical for virulence of African trypanosomes.

African trypanosomes, Trypanosoma brucei spp., are lethal pathogens that cause substantial human suffering and limit economic development in some of the world's most impoverished regions. The name Trypanosoma ("auger cell") derives from the parasite's distinctive motility, which is driven by a single flagellum. However, despite decades of study, a requirement for trypanosome motility in mammalian host infection has not been established. LC1 is a conserved dynein subunit required for flagellar motility. Prior studies with a conditional RNAi-based LC1 mutant, RNAi-K/R, revealed that parasites with defective motility could infect mice. However, RNAi-K/R retained residual expression of wild-type LC1 and residual motility, thus precluding definitive interpretation. To overcome these limitations, here we generate constitutive mutants in which both LC1 alleles are replaced with mutant versions. These double knock-in mutants show reduced motility compared to RNAi-K/R and are viable in culture, but are unable to maintain bloodstream infection in mice. The virulence defect is independent of infection route but dependent on an intact host immune system. By comparing different mutants, we also reveal a critical dependence on the LC1 N-terminus for motility and virulence. Our findings demonstrate that trypanosome motility is critical for establishment and maintenance of bloodstream infection, implicating dynein-dependent flagellar motility as a potential drug target

Endemic Human Monkeypox, Democratic Republic of Congo, 2001–2004

By analyzing vesicle fluids and crusted scabs from 136 persons with suspected monkeypox, we identified 51 cases of monkeypox by PCR, sequenced the hemagglutinin gene, and confirmed 94% of cases by virus culture. PCR demonstrated chickenpox in 61 patients. Coinfection with both viruses was found in 1 additional patient

Using Remote Sensing to Map the Risk of Human Monkeypox Virus in the Congo Basin

Although the incidence of human monkeypox has greatly increased in Central Africa over the last decade, resources for surveillance remain extremely limited. We conducted a geospatial analysis using existing data to better inform future surveillance efforts. Using active surveillance data collected between 2005 and 2007, we identified locations in Sankuru district, Democratic Republic of Congo (DRC) where there have been one or more cases of human monkeypox. To assess what taxa constitute the main reservoirs of monkeypox, we tested whether human cases were associated with (i) rope squirrels (Funisciurus sp.), which were implicated in monkeypox outbreaks elsewhere in the DRC in the 1980s, or (ii) terrestrial rodents in the genera Cricetomys and Graphiurus, which are believed to be monkeypox reservoirs in West Africa. Results suggest that the best predictors of human monkeypox cases are proximity to dense forests and associated habitat preferred by rope squirrels. The risk of contracting monkeypox is significantly greater near sites predicted to be habitable for squirrels (OR = 1.32; 95% CI 1.08–1.63). We recommend that semi-deciduous rainforests with oil-palm, the rope squirrel’s main food source, be prioritized for monitoring

Role of Flagellar Motility in Trypanosoma brucei Pathogenesis

The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that drives parasite motility and is receiving increased attention as a potential drug target. Parasite motility is suspected to contribute to infection and disease pathogenesis in the mammalian host. However, it has not been possible to test this hypothesis owing to lack of motility mutants that are viable in the bloodstream life cycle stage that infects the mammalian host. In the first part of my dissertation we identified a viable bloodstream-form motility mutant in 427-derived T. brucei and by adapting published approaches we set up mouse infection models of African trypanosomiasis. To assess the impact of trypanosome motility on infection in mice we used these mutants in a mouse infection model and showed that disrupting parasite motility has no discernible effect on T. brucei bloodstream infection. This presents the first ever investigation of the influence of parasite motility on infection of the mammalian host. Mutant cells used were derived from the laboratory-adapted strain 427-BSSM that causes an acute infection that progress rapidly in mice. This quick disease progression limits any reliable assessment of the CNS penetration, which commonly takes more than 14 days. To allow direct investigation of the requirement of parasite motility in the central nervous system (CNS) invasion, we have generated motility mutants in T. brucei strains that cause chronic infection in mice. Identification of motility mutants in pleomorphic BSF T. brucei that causes chronic infection will now make it possible for the first time to test if parasite motility is required for CNS penetration. Traditionally, assessment of T. brucei infection is based upon examining parasitemia in blood and limited use of histochemistry to determine parasite presence in chemically-treated tissues. We have developed an advanced live-cell imaging approach using fluorescent T. brucei that will facilitate detailed dynamic studies of infection. This system enabled visualization of T. brucei ex vivo in mouse tissues as well as in vivo in whole live zebrafish embryos. Further validation of mCherry parasites at a microscale level revealed trypanosomes at single-cell resolution in ex vivo mouse tissues and in blood vessels of live fish. Hence, these systems have the potential for uncovering novel features of host-parasite interactions that could lead to drug, vaccine and diagnostics development, all of which is expected to ameliorate patient management in sleeping sickness. A major roadblock to the study of the flagellum is a lack of facile methods for systematic mutational analysis of flagellar genes. We recently established systems for structure-function analysis of proteins in T. brucei, which has emerged as an excellent model to study the eukaryotic flagellum. Several flagellar proteins have been identified but there is scant information on molecular mechanisms underlying these proteins functions, individually or collectively. For instance, key amino acids and domains required for these proteins are for the most part unknown. To exploit the flagellum as a drug target it is crucial that we deepen understanding of molecular mechanisms of flagellum protein function. To start bridging this gap, we applied our structure-function system to define amino acids required for IFT88 and trypanin protein function in flagellum assembly and motility. Our studies tested amino acids that correspond to IFT88 mutations observed in human patients with defective cilia and showed which of these mutations are loss of function mutations versus polymorphisms. We have also uncovered key domains essential for the assembly and function of the dynein regulatory protein trypanin. Altogether, these investigations broadly contribute to understanding T. brucei pathogenesis mechanisms and expand our knowledge of flagellum motility functions in trypanosomes, which are directly applicable to other flagellated protozoan parasites. In humans, the flagellum, also called a cilium, is required for normal development and physiology and genetic changes in flagellar genes cause many human heritable diseases. Thus, our studies are also relevant to eukaryotic cell biology in connection to human health and disease

Role of Flagellar Motility in Trypanosoma brucei Pathogenesis

The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that drives parasite motility and is receiving increased attention as a potential drug target. Parasite motility is suspected to contribute to infection and disease pathogenesis in the mammalian host. However, it has not been possible to test this hypothesis owing to lack of motility mutants that are viable in the bloodstream life cycle stage that infects the mammalian host. In the first part of my dissertation we identified a viable bloodstream-form motility mutant in 427-derived T. brucei and by adapting published approaches we set up mouse infection models of African trypanosomiasis. To assess the impact of trypanosome motility on infection in mice we used these mutants in a mouse infection model and showed that disrupting parasite motility has no discernible effect on T. brucei bloodstream infection. This presents the first ever investigation of the influence of parasite motility on infection of the mammalian host. Mutant cells used were derived from the laboratory-adapted strain 427-BSSM that causes an acute infection that progress rapidly in mice. This quick disease progression limits any reliable assessment of the CNS penetration, which commonly takes more than 14 days. To allow direct investigation of the requirement of parasite motility in the central nervous system (CNS) invasion, we have generated motility mutants in T. brucei strains that cause chronic infection in mice. Identification of motility mutants in pleomorphic BSF T. brucei that causes chronic infection will now make it possible for the first time to test if parasite motility is required for CNS penetration. Traditionally, assessment of T. brucei infection is based upon examining parasitemia in blood and limited use of histochemistry to determine parasite presence in chemically-treated tissues. We have developed an advanced live-cell imaging approach using fluorescent T. brucei that will facilitate detailed dynamic studies of infection. This system enabled visualization of T. brucei ex vivo in mouse tissues as well as in vivo in whole live zebrafish embryos. Further validation of mCherry parasites at a microscale level revealed trypanosomes at single-cell resolution in ex vivo mouse tissues and in blood vessels of live fish. Hence, these systems have the potential for uncovering novel features of host-parasite interactions that could lead to drug, vaccine and diagnostics development, all of which is expected to ameliorate patient management in sleeping sickness. A major roadblock to the study of the flagellum is a lack of facile methods for systematic mutational analysis of flagellar genes. We recently established systems for structure-function analysis of proteins in T. brucei, which has emerged as an excellent model to study the eukaryotic flagellum. Several flagellar proteins have been identified but there is scant information on molecular mechanisms underlying these proteins functions, individually or collectively. For instance, key amino acids and domains required for these proteins are for the most part unknown. To exploit the flagellum as a drug target it is crucial that we deepen understanding of molecular mechanisms of flagellum protein function. To start bridging this gap, we applied our structure-function system to define amino acids required for IFT88 and trypanin protein function in flagellum assembly and motility. Our studies tested amino acids that correspond to IFT88 mutations observed in human patients with defective cilia and showed which of these mutations are loss of function mutations versus polymorphisms. We have also uncovered key domains essential for the assembly and function of the dynein regulatory protein trypanin. Altogether, these investigations broadly contribute to understanding T. brucei pathogenesis mechanisms and expand our knowledge of flagellum motility functions in trypanosomes, which are directly applicable to other flagellated protozoan parasites. In humans, the flagellum, also called a cilium, is required for normal development and physiology and genetic changes in flagellar genes cause many human heritable diseases. Thus, our studies are also relevant to eukaryotic cell biology in connection to human health and disease

Trypanosome flagellum motility and infection

The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that drives parasite motility and is receiving increased attention as a potential drug target. In the mammalian host, parasite motility is suspected to contribute to infection and disease pathogenesis. However, it has not been possible to test this hypothesis owing to lack of motility mutants that are viable in the bloodstream life cycle stage that infects the mammalian host. We recently identified a bloodstream-form motility mutant in 427-derived T. brucei in which point mutations in the LC1 dynein subunit disrupt propulsive motility but do not affect viability. These mutants have an actively beating flagellum, but cannot translocate. Here we demonstrate that the LC1 point mutant fails to show enhanced cell motility upon increasing viscosity of the surrounding medium, which is a hallmark of wild type T. brucei, thus indicating that motility of the mutant is fundamentally altered compared with wild type cells. We next used the LC1 point mutant to assess the influence of trypanosome motility on infection in mice. Wesurprisingly found that disrupting parasite motility has no discernible effect on T. brucei bloodstream infection. Infection time-course, maximum parasitaemia, number of waves of parasitaemia, clinical features and disease outcome are indistinguishable between motility mutant and control parasites. Our studies provide an important step toward understanding the contribution of parasite motility to infection and a foundation for future investigations of T. brucei interaction with the mammalian host

Parasite motility is critical for virulence of African trypanosomes.

African trypanosomes, Trypanosoma brucei spp., are lethal pathogens that cause substantial human suffering and limit economic development in some of the world's most impoverished regions. The name Trypanosoma ("auger cell") derives from the parasite's distinctive motility, which is driven by a single flagellum. However, despite decades of study, a requirement for trypanosome motility in mammalian host infection has not been established. LC1 is a conserved dynein subunit required for flagellar motility. Prior studies with a conditional RNAi-based LC1 mutant, RNAi-K/R, revealed that parasites with defective motility could infect mice. However, RNAi-K/R retained residual expression of wild-type LC1 and residual motility, thus precluding definitive interpretation. To overcome these limitations, here we generate constitutive mutants in which both LC1 alleles are replaced with mutant versions. These double knock-in mutants show reduced motility compared to RNAi-K/R and are viable in culture, but are unable to maintain bloodstream infection in mice. The virulence defect is independent of infection route but dependent on an intact host immune system. By comparing different mutants, we also reveal a critical dependence on the LC1 N-terminus for motility and virulence. Our findings demonstrate that trypanosome motility is critical for establishment and maintenance of bloodstream infection, implicating dynein-dependent flagellar motility as a potential drug target

Cytokine modulation correlates with severity of monkeypox disease in humans.

BackgroundHuman monkeypox is a zoonotic disease endemic to parts of Africa. Similar to other orthopoxviruses, virus and host have considerable interactions through immunomodulation. These interactions likely drive the establishment of a productive infection and disease progression, resulting in the range of disease presentations and case fatality rates observed for members of the Orthopoxvirus genus.ObjectivesMuch of our understanding about the immune response to orthopoxvirus infection comes from either in vitro or in vivo studies performed in small animals or non-human primates. Here, we conducted a detailed assessment of cytokine responses to monkeypox virus using serum from acutely ill humans collected during monkeypox active disease surveillance (2005-2007) in the Democratic Republic of the Congo.Study designNineteen serum samples that were from patients with confirmed monkeypox virus infections were selected for cytokine profiling. Cytokine profiling was performed on the Bio-Rad Bioplex 100 system using a 30-plex human cytokine panel.ResultsCytokine profiling revealed elevated cytokine concentrations in all samples. Overproduction of certain cytokines (interleukin [IL]-2R, IL-10, and granulocyte macrophage-colony stimulating factor were observed in patients with serious disease (defined as >250 lesions based on the World Health Organization scoring system).ConclusionsThe data suggest that cytokine modulation affects monkeypox disease severity in humans