In vivo growth of Staphylococcus lugdunensis is facilitated by the concerted function of heme and non-heme iron acquisition mechanisms

Acquisition of iron underpins the ability of pathogens to cause disease and Staphylococcus lugdunensis has increasingly been recognized as a pathogen that can cause serious infection. In this study, we sought to address the knowledge gap that exists regarding the iron acquisition mechanisms employed by S. lugdunensis, especially during infection of the mammalian host. Here we show that S. lugdunensis utilizes diverse genome encoded iron acquisition mechanisms to satisfy its need for this nutrient. Indeed, S. lugdunensis can usurp hydroxamate siderophores, and staphyloferrin A and B from S. aureus, using the fhuC ATPase-encoding gene. Acquisition of catechol siderophores and catecholamine stress hormones necessitates the presence of the sst-1 transporter-encoding locus, but not the sst-2 locus. Iron-dependent growth in acidic culture conditions necessitates the feoAB locus. Heme iron is acquired via expression of the iron-regulated surface determinant (isd) locus. During systemic infection of mice we demonstrate that while S. lugdunensis does not cause overt illness, it does colonize and proliferate to high numbers in the kidneys. By combining mutations in the various iron acquisition loci, we further demonstrate that only a strain mutated for all of isd, fhuC, sst-1, and feo, versus combination mutants carrying wild type copies of any one of those loci, was attenuated in its ability to proliferate to high numbers in kidneys. Taken together our data reveal that S. lugdunensis requires a repertoire of both heme and non-heme iron acquisition mechanisms to proliferate during systemic infection of mammals

Comparative transcriptome profiles of Schistosoma japonicum larval stages: Implications for parasite biology and host invasion

Schistosoma japonicum is prevalent in Asia with a wide mammalian host range, which leads to highly harmful zoonotic parasitic diseases. Most previous transcriptomic studies have been performed on this parasite, but mainly focus on stages inside the mammalian host. Moreover, few larval transcriptomic data are available in public databases. Here we mapped the detailed transcriptome profiles of four S. japonicum larval stages including eggs, miracidia, sporocysts and cercariae, providing a comprehensive development picture outside of the mammalian host. By analyzing the stage-specific/enriched genes, we identified functional genes associated with the biological characteristic at each stage: e.g. we observed enrichment of genes necessary for DNA replication only in sporocysts, while those involved in proteolysis were upregulated in sporocysts and/or cercariae. This data indicated that miracidia might use leishmanolysin and neprilysin to penetrate the snail, while elastase (SjCE2b) and leishmanolysin might contribute to the cercariae invasion. The expression profile of stem cell markers revealed potential germinal cell conversion during larval development. Additionally, our analysis indicated that tandem duplications had driven the expansion of the papain family in S. japonicum. Notably, all the duplicated cathepsin B-like proteases were highly expressed in cercariae. Utilizing our 3rd version of S. japonicum genome, we further characterized the alternative splicing profiles throughout these four stages. Taken together, the present study provides compressive gene expression profiles of S. japonicum larval stages and identifies a set of genes that might be involved in intermediate and definitive host invasion.

The Occurrence of Malignancy in Trypanosoma brucei brucei by Rapid Passage in Mice

Pleomorphic Trypanosoma brucei are best known for their tightly controlled cell growth and developmental program, which ensures their transmissibility and host fitness between the mammalian host and insect vector. However, after long-term adaptation in the laboratory or by natural evolution, monomorphic parasites can be derived. The origin of these monomorphic forms is currently unclear. Here, we produced a series of monomorphic trypanosome stocks by artificially syringe-passage in mice, creating snapshots of the transition from pleomorphism to monomorphism. We then compared these artificial monomorphic trypanosomes, alongside several naturally monomorphic T. evansi and T. equiperdum strains, with the pleomorphic T. brucei. In addition to failing to generate stumpy forms in animal bloodstream, we found that monomorphic trypanosomes from laboratory and nature exhibited distinct differentiation patterns, which are reflected by their distinct differentiation potential and transcriptional changes. Lab-adapted monomorphic trypanosomes could still be induced to differentiate, and showed only minor transcriptional differences to that of the pleomorphic slender forms but some accumulated differences were observed as the passages progress. All naturally monomorphic strains completely fail to differentiate, corresponding to their impaired differentiation regulation. We propose that the natural phenomenon of trypanosomal monomorphism is actually a malignant manifestation of protozoal cells. From a disease epidemiological and evolutionary perspective, our results provide evidence for a new way of thinking about the origin of these naturally monomorphic strains, the malignant evolution of trypanosomes may raise some concerns. Additionally, these monomorphic trypanosomes may reflect the quantitative and qualitative changes in the malignant evolution of T. brucei, suggesting that single-celled protozoa may also provide the most primitive model of cellular malignancy, which could be a primitive and inherent biological phenomenon of eukaryotic organisms from protozoans to mammals.

Genomic occupancy of the bromodomain protein Bdf3 is dynamic during differentiation of African trypanosomes from bloodstream to procyclic forms

Trypanosoma brucei , the causative agent of Human and Animal African trypanosomiasis, cycles between a mammalian host and a tsetse fly vector. The parasite undergoes huge changes in morphology and metabolism as it adapts to each host environment. These changes are reflected in the differing transcriptomes of parasites living in each host. While changes in the transcriptome have been well catalogued for parasites differentiating from the mammalian bloodstream to the insect stage, it remains unclear whether chromatin interacting proteins mediate transcriptomic changes during life cycle adaptation. We and others have shown that chromatin interacting bromodomain proteins localize to transcription start sites in bloodstream parasites, but whether the localization of bromodomain proteins changes as parasites differentiate from bloodstream to insect stage parasites remains unknown. To address this question, we performed Cleavage Under Target and Release Using Nuclease (CUT&RUN) timecourse experiments using a tagged version of Bromodomain Protein 3 (Bdf3) in parasites differentiating from bloodstream to insect stage forms. We found that Bdf3 occupancy at most loci increased at 3 hours following onset of differentiation and decreased thereafter. A number of sites with increased bromodomain protein occupancy lie proximal to genes known to have altered transcript levels during differentiation, such as procyclins, procyclin associated genes, and invariant surface glycoproteins. While most Bdf3 occupied sites are observed throughout differentiation, a very small number appear de novo as differentiation progresses. Notably, one such site lies proximal to the procyclin gene locus, which contains genes essential for remodeling surface proteins following transition to the insect stage. Overall, these studies indicate that occupancy of chromatin interacting proteins is dynamic during life cycle stage transitions, and provides the groundwork for future studies aimed at uncovering whether changes in bromodomain protein occupancy affect transcript levels of neighboring genes. Additionally, the optimization of CUT&RUN for use in Trypanosoma brucei may prove helpful for other researchers as an alternative to Chromatin Immunoprecipitation (ChIP).

The genomic basis of host and vector specificity in non-pathogenic trypanosomatids

The ability of trypanosome parasites to survive and sustain infections is dependent on diverse and intricate immune evasion mechanisms. Pathogenic trypanosomes often have broad host niches that preclude identification of host specific adaptations. In contrast, some non-pathogenic species of the genus Trypanosoma have highly specific hosts and vectors. Trypanosoma theileri, a non-pathogenic parasite of bovines, has a predicted surface protein architecture that likely aids survival in its mammalian host, distinct from the dominant variant surface glycoprotein coat of pathogenic African trypanosomes. In both species, their surface proteins are encoded by genes which account for ~10% of their genome. A non-pathogenic parasite of sheep, Trypanosoma melophagium, is transmitted by the sheep ked and is closely related to T. theileri. To explore host and vector specificity between these closely related species, we sequenced the T. melophagium genome and transcriptome and an annotated draft genome was assembled. T. melophagium was compared to 43 kinetoplastid genomes, including T. theileri. T. melophagium and T. theileri have an AT biased genome, the greatest bias of publicly available trypanosomatids. This trend may result from selection acting to decrease the genome nucleotide cost. The T. melophagium genome is 6.3Mb smaller than T. theileri and large families of proteins, characteristic of the predicted surface of T. theileri, were found to be absent or greatly reduced in T. melophagium. Instead, T. melophagium has modestly expanded protein families associated with the avoidance of complement-mediated lysis. The genome of T. melophagium contains core genes required for development, glycolysis, RNA interference, and meiotic exchange, each being shared with T. theileri. Comparisons between T. melophagium and T. theileri provide insight into the specific adaptations of these related trypanosomatids to their distinct mammalian hosts and arthropod vectors.

Identification of the Host Substratome of Leishmania-Secreted Casein Kinase 1 Using a SILAC-Based Quantitative Mass Spectrometry Assay

Leishmaniasis is a severe public health problem, caused by the protozoan Leishmania. This parasite has two developmental forms, extracellular promastigote in the insect vector and intracellular amastigote in the mammalian host where it resides inside the phagolysosome of macrophages. Little is known about the virulence factors that regulate host-pathogen interactions and particularly host signalling subversion. All the proteomes of Leishmania extracellular vesicles identified the presence of Leishmania casein kinase 1 (L-CK1.2), a signalling kinase. L-CK1.2 is essential for parasite survival and thus might be essential for host subversion. To get insights into the functions of L-CK1.2 in the macrophage, the systematic identification of its host substrates is crucial, we thus developed an easy method to identify substrates, combining phosphatase treatment, in vitro kinase assay and Stable Isotope Labelling with Amino acids in Cell (SILAC) culture-based mass spectrometry. Implementing this approach, we identified 225 host substrates as well as a potential novel phosphorylation motif for CK1. We confirmed experimentally the enrichment of our substratome in bona fide L-CK1.2 substrates and showed they were also phosphorylated by human CK1δ. L-CK1.2 substratome is enriched in biological processes such as “viral and symbiotic interaction,” “actin cytoskeleton organisation” and “apoptosis,” which are consistent with the host pathways modified by Leishmania upon infection, suggesting that L-CK1.2 might be the missing link. Overall, our results generate important mechanistic insights into the signalling of host subversion by these parasites and other microbial pathogens adapted for intracellular survival.

High genome plasticity and frequent genetic exchange in Leishmania tropica isolates from Afghanistan, Iran and Syria

Background The kinetoplastid protozoan Leishmania tropica mainly causes cutaneous leishmaniasis in humans in the Middle East, and relapse or treatment failure after treatment are common in this area. L. tropica’s digenic life cycle includes distinct stages in the vector sandfly and the mammalian host. Sexual reproduction and genetic exchange appear to occur more frequently than in other Leishmania species. Understanding these processes is complicated by chromosome instability during cell division that yields aneuploidy, recombination and heterozygosity. This combination of rare recombination and aneuploid permits may reveal signs of hypothetical parasexual mating, where diploid cells fuse to form a transient tetraploid that undergoes chromosomal recombination and gradual chromosomal loss. Methodology/principal findings The genome-wide SNP diversity from 22 L. tropica isolates showed chromosome-specific runs of patchy heterozygosity and extensive chromosome copy number variation. All these isolates were collected during 2007–2017 in Sweden from patients infected in the Middle East and included isolates from a patient possessing two genetically distinct leishmaniasis infections three years apart with no evidence of re-infection. We found differing ancestries on the same chromosome (chr36) across multiple samples: matching the reference genome with few derived alleles, followed by blocks of heterozygous SNPs, and then by clusters of homozygous SNPs with specific recombination breakpoints at an inferred origin of replication. Other chromosomes had similar marked changes in heterozygosity at strand-switch regions separating polycistronic transcriptional units. Conclusion/significance These large-scale intra- and inter-chromosomal changes in diversity driven by recombination and aneuploidy suggest multiple mechanisms of cell reproduction and diversification in L. tropica, including mitotic, meiotic and parasexual processes. It underpins the need for more genomic surveillance of Leishmania, to detect emerging hybrids that could spread more widely and to better understand the association between genetic variation and treatment outcome. Furthering our understanding of Leishmania genome evolution and ancestry will aid better diagnostics and treatment for cutaneous leishmaniasis caused by L.tropica in the Middle East.

Metaproteomic Profiling of Fungal Gut Colonization in Gnotobiotic Mice

Background: Eukaryotic microbes can modulate mammalian host health and disease states, yet the molecular contribution of gut fungi remains nascent. We previously showed that mice exclusively colonised with fungi displayed increased sensitivity to allergic airway inflammation and had fecal metabolite profiles similar to germ-free mice. This marginal effect on the host metabolome suggested that fungi do not primarily use metabolites to modulate the host immune system.Methods: To describe functional changes attributed to fungal colonisation, we performed mass spectrometry-based analyses of feces (Label-Free Quantitative; LFQ) and the small intestine (labeling with Tandem Mass Tag; TMT) of gnotobiotic mice colonised with defined consortia of twelve bacterial species, five fungal species, or both. We also evaluated the effect of microbiome perturbances on the metaproteome by analysing feces from mouse pups treated with an antibiotic or antifungal.Results: We detected 6,675 proteins in the mice feces, of which 3,845 had determined LFQ levels. Analysis of variance showed changes in the different gnotobiotic mouse groups; specifically, 46% of 2,860 bacterial, 15% of 580 fungal, and 76% of 405 mouse quantified proteins displayed differential levels. The antimicrobial treatments resulted in lasting changes in the bacterial and fungal proteomes, suggesting that the antimicrobials impacted the entire community. Fungal colonisation resulted in changes in host proteins functional in innate immunity as well as metabolism, predicting specific roles of gut fungi on host systems during early developmental stages. Several of the detected fungal proteins (3% of 1,492) have been previously reported as part of extracellular vesicles and having immunomodulating properties. Using an isobaric labelling TMT approach for profiling low abundant proteins of the jejunal tissue, we confirmed that the five fungal species differentially impacted the host intestinal proteome compared to the bacterial consortium. The detected changes in mouse jejunal proteins (4% of 1,514) were mainly driven by metabolic proteins. Conclusions: We used quantitative proteomic profiling of gnotobiotic conditions to show how colonisation with selected fungal species impacts the host gut proteome. Our results suggest that an increased abundance of certain gut fungal species in early life may affect the developing intracellular attributes of epithelial and immune cells.

Presence of Conchoderma auritum and C. virgatum (Maxillopoda, Lepadidae) and Gloiopotes huttoni (Copepoda, Caligidae) on the black marlin, from the Gulf of Tehuantepec, Mexico

This is the first report of coexistence of pedunculate barnacles, Conchoderma auritum and C. virgatum in the mouth, and with the copepod Gloiopotes huttoni in the precaudal zone, on the black marlin, Istiompax indica, a as host. This is also the first note of a new non-mammalian host for C. auritum. The host specimen was caught in sport fishing in the Gulf of Tehuantepec, eastern Pacific. A review of registered occurrences of the genus Conchoderma in species of the fish family Istiophoridae is provided.