Background: Loss of virulence is a phenotypic adaptation commonly seen in prokaryotic and eukaryotic pathogens. This mechanism is not well studied, especially in organisms with multiple host and life cycle stages such as Babesia, a tick-transmitted hemoparasite of humans and animals. B. bovis, which infects cattle, has naturally occurring virulent strains that can be reliably attenuated in vivo. Previous studies suggest the virulence loss mechanism may involve post-genomic modification. We investigated the transcriptome profiles of two geographically distinct B. bovis virulent and attenuated strain pairs to better understand virulence loss and to gain insight into pathogen adaptation strategies. Results: Expression microarray and RNA-sequencing approaches were employed to compare transcriptome profiles of two B. bovis strain pairs, with each pair consisting of a virulent parental and its attenuated derivative strain. Differentially regulated transcripts were identified within each strain pair. These included genes encoding for VESA1, SmORFs, undefined membrane and hypothetical proteins. The majority of individual specific gene transcripts differentially regulated within a strain were not shared between the two strains. There was a disproportionately greater number of ves genes upregulated in the virulent parental strains. When compared with their attenuated derivatives, divergently oriented ves genes were included among the upregulated ves genes in the virulent strains, while none of the upregulated ves genes in the attenuated derivatives were oriented head to head. One gene family whose specific members were consistently and significantly upregulated in expression in both attenuated strains was spherical body protein (SBP) 2 encoding gene where SBP2 truncated copies 7, 9 and 11 transcripts were all upregulated. Conclusions: We conclude that ves heterodimer pair upregulation and overall higher frequency of ves gene expressions in the virulent strains is consistent with the involvement of this gene family in virulence. This is logical given the role of VESA1 proteins in cytoadherence of infected cells to endothelial cells. However, upregulation of some ves genes in the attenuated derivatives suggests that the consequence of upregulation is gene-specific. Furthermore, upregulation of the spherical body protein 2 gene family may play a role in the attenuated phenotype. Exactly how these two gene families may contribute to the loss or gain of virulence is discussed.

The obligate intracellular parasite Toxoplasma gondii is auxotrophic for several key metabolites and must scavenge these from the host. It is unclear how T. gondii manipulates host metabolism to support its overall growth rate and non-essential metabolites. To investigate this question, we measured changes in the joint host-parasite metabolome over a time course of infection. Host and parasite transcriptomes were simultaneously generated to determine potential changes in expression of metabolic enzymes. T. gondii infection changed metabolite abundance in multiple metabolic pathways, including the tricarboxylic acid cycle, the pentose phosphate pathway, glycolysis, amino acid synthesis, and nucleotide metabolism. Our analysis indicated that changes in some pathways, such as the tricarboxylic acid cycle, were mirrored by changes in parasite transcription, while changes in others, like the pentose phosphate pathway, were paired with changes in both the host and parasite transcriptomes. Further experiments led to the discovery of a T. gondii enzyme, sedoheptulose bisphosphatase, which funnels carbon from glycolysis into the pentose phosphate pathway through an energetically driven dephosphorylation reaction. This additional route for ribose synthesis appears to resolve the conflict between the T. gondii tricarboxylic acid cycle and pentose phosphate pathway, which are both NADP+ dependent. Sedoheptulose bisphosphatase represents a novel step in T. gondii central carbon metabolism that allows T. gondii to energetically-drive ribose synthesis without using NADP+. The obligate intracellular parasite T. gondii is commonly found among human populations worldwide and poses severe health risks to fetuses and individuals with AIDS. While some treatments are available they are limited in scope. A possible target for new therapies is T. gondii’s incomplete metabolism, which makes it heavily reliant on its host. In this study, we generated a joint host/parasite metabolome to better understand host manipulation by the parasite and to discover unique aspects of T. gondii metabolism that could serve as the next generation of drug targets. Metabolomic analysis of T. gondii infection over time found broad alterations to host metabolism by the parasite in both energetic and biosynthetic pathways. We discovered a new T. gondii enzyme, sedoheptulose bisphosphatase, which redirects carbon from glycolysis into the pentose phosphate pathway. The wholesale remodeling of host metabolism for optimal parasite growth is also of interest, although the mechanisms behind this host manipulation must be further studied before therapeutic targets can be identified.

Apicomplexans are a diverse group of obligate parasites occupying different intracellular niches that require modification to meet the needs of the parasite. To efficiently manipulate their environment, apicomplexans translocate numerous parasite proteins into the host cell. Whereas some parasites remain contained within a parasitophorous vacuole membrane (PVM) throughout their developmental cycle, others do not, a difference that affects the machinery needed for protein export. A signal‐mediated pathway for protein export into the host cell has been characterized in Plasmodium parasites, which maintain the PVM. Here, we functionally demonstrate an analogous host‐targeting pathway involving organellar staging prior to secretion in the related bovine parasite, Babesia bovis, a parasite that destroys the PVM shortly after invasion. Taking into account recent identification of a similar signal‐mediated pathway in the coccidian parasite Toxoplasma gondii, we suggest a model in which this conserved pathway has evolved in multiple steps from signal‐mediated trafficking to specific secretory organelles for controlled secretion to a complex protein translocation process across the PVM.

Bovine babesiosis caused by Babesia bovis is a tick-borne hemoparasitic disease of global impact, and improved control is needed. In B. bovis, spherical body protein 2 (SBP-2) truncated copies 7, 9 and 11 (sbp2t7, sbp2t9 and sbp2t11) gene transcripts were recently reported to be significantly upregulated in two geographically distinct attenuated B. bovis strains. In the present work, additional virulent and attenuated B. bovis strain pairs were compared in order to corroborate this finding. Sequences of the sbp2t7, sbp2t9 and sbp2t11 genes were not fully conserved among geographically distinct B. bovis strains, and varied between 70.6–93.3% sequence identity in all three genes. Comparisons among transcript levels of the three sbp2t genes of distinct virulent-attenuated B. bovis strain pairs confirmed that upregulation of the sbp2t11 gene was exclusively associated with an attenuated phenotype in the studied strain pairs. This rejects sbp2t7 and sbp2t9 as reliable attenuation markers. In addition, SBP2t11 protein was found to be significantly overexpressed in Texas attenuated B. bovis in comparison to the Texas virulent strain. Finally, sbp2t11 was differentially expressed in blood stages of the parasite but undetectable in Texas strain kinetes. Sbp2t11 is a strong candidate as a reliable attenuation marker for B. bovis, based on its consistent pattern of upregulation in four distinct attenuated strains when compared to their virulent parental strains. Sbp2t11 may only have functional roles associated with erythrocyte infection. Identification of attenuation markers will lead to future research focused on the production of novel and safer subunit and genetically defined vaccines against B. bovis.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular parasite that infects warm-blooded vertebrates across the world. In humans, seropositivity rates of T. gondii range from 10% to 90% across communities. Despite its prevalence, few studies address how T. gondii infection changes the metabolism of host cells. In this study, we investigate how T. gondii manipulates the host cell metabolic environment by monitoring the metabolic response over time using noninvasive autofluorescence lifetime imaging of single cells, metabolite analysis, extracellular flux analysis, and reactive oxygen species (ROS) production. Autofluorescence lifetime imaging indicates that infected host cells become more oxidized and have an increased proportion of bound NAD(P)H compared to uninfected controls. Over time, infected cells also show decreases in levels of intracellular glucose and lactate, increases in oxygen consumption, and variability in ROS production. We further examined changes associated with the pre-invasion “kiss and spit” process using autofluorescence lifetime imaging, which also showed a more oxidized host cell with an increased proportion of bound NAD(P)H over 48 hours compared to uninfected controls, suggesting that metabolic changes in host cells are induced by T. gondii kiss and spit even without invasion. IMPORTANCE: This study sheds light on previously unexplored changes in host cell metabolism induced by T. gondii infection using noninvasive, label-free autofluorescence imaging. In this study, we use optical metabolic imaging (OMI) to measure the optical redox ratio (ORR) in conjunction with fluorescence lifetime imaging microscopy (FLIM) to noninvasively monitor single host cell response to T. gondii infection over 48 hours. Collectively, our results affirm the value of using autofluorescence lifetime imaging to noninvasively monitor metabolic changes in host cells over the time course of a microbial infection. Understanding this metabolic relationship between the host cell and the parasite could uncover new treatment and prevention options for T. gondii infections worldwide.