Elsevier

Experimental Parasitology

Volume 187, April 2018, Pages 75-85
Experimental Parasitology

Modulation of cis- and trans- Golgi and the Rab9A-GTPase during infection by Besnoitia besnoiti, Toxoplasma gondii and Neospora caninum

https://doi.org/10.1016/j.exppara.2018.02.008Get rights and content

Highlights

  • B. besnoiti infection compacts the host cell Golgi.

  • T. gondii infection fragments the host cell Golgi.

  • N. caninum infection does not influence host cell's Golgi morphology.

  • The three parasites recruit host cell Rab9A-associated vesicles to the PV.

  • T. gondii induces an increased expression of Rab9A mRNA and protein in invaded cells.

Abstract

Like most intracellular pathogens, the apicomplexan parasites Besnoitia besnoiti, Toxoplasma gondii and Neospora caninum scavenge metabolites from their host cells. Recruitment of the Golgi complex to the vicinity of the parasitophorous vacuole (PV) is likely to aid in this process. In this work, we comparatively assessed B. besnoiti, T. gondii and N. caninum infected human retinal pigmented epithelial (hTERT-RPE-1) cells at 24 h post-infection and used antibodies to confirm Golgi ribbon compaction in B. besnoiti, and Golgi ribbon dispersion in T. gondii, while no alteration in Golgi morphology was seen in N. caninum infected cells. In either case, the Golgi stacks of infected cells contained both cis- (GM130) and trans- (TGN46) Golgi proteins. The localization of Rab9A, an important regulator of endosomal trafficking, was also studied. GFP-tagged Rab9A was recruited to the vicinity of the PV of all three parasites. Toxoplasma-infected cells exhibited increased expression of Rab9A in comparison to non-infected cells. However, Rab9A expression levels remained unaltered upon infection with N. caninum and B. besnoiti tachyzoites. In contrast to Rab9A, a GFP-tagged dominant negative mutant form of Rab9A (Rab9A DN), was not recruited to the PV, and the expression of Rab9A DN did not affect host cell invasion nor replication by all three parasites. Thus, B. besnoiti, T. gondii and N. caninum show similarities but also differences in how they affect constituents of the endosomal/secretory pathways.

Introduction

The protozoans Besnoitia besnoiti, Toxoplasma gondii and Neospora caninum are closely related apicomplexan parasites, and they are the etiological agents of besnoitiosis, toxoplasmosis, and neosporosis, respectively. These parasites are of veterinary importance, and T. gondii is also of high medical relevance, especially during pregnancy and in immunocompromised individuals. All three diseases cause significant economic losses in livestock industry: in bovine besnoitiosis, chronic infection causes low body condition, scleroderma, pronounced thickening of the limbs, and difficult and painful locomotion (Pols, 1960); toxoplasmosis is one of the major causes of abortion in sheep (Innes, 2010); and neosporosis is an important cause of bovine abortion, stillbirth, and birth of weak calves (Dubey et al., 2017).

Similar to other obligate intracellular pathogens, B. besnoiti, T. gondii, and N. caninum have an impact on the intracellular organization of their host cells. For instance, infection results in remodeling of the host cell cytoskeleton, with repositioning of the centrosome, mitochondria, endoplasmic reticulum (ER) and Golgi complex (Coppens et al., 2006; Wang et al., 2010; Romano et al., 2013; Cardoso et al., 2014, 2016; Pernas et al., 2014; Nolan et al., 2015). It is believed that parasites benefit from this, as it facilitates interference in signaling pathways, which in turn can result in evasion of the host immune responses. In addition, cytoskeletal remodeling enables apicomplexans to scavenge essential host cell nutrients. Considering that the Golgi complex receives newly synthesized proteins and lipids from the endoplasmic reticulum, it is conceivable that recruitment of this organelle to the vicinity of the parasitophorous vacuole (PV) facilitates access to essential host cell components. In B. besnoiti, T. gondii, and N. caninum infected host cells the Golgi complex is located close to the PV. However, B. besnoiti induces Golgi complex compaction; T. gondii causes fragmentation; and N. caninum does not seem to have a major impact on host cell Golgi morphology (Walker et al., 2008; Romano et al., 2013; Cardoso et al., 2014; Nolan et al., 2015).

The Golgi complex morphology remodeling observed during and after B. besnoiti and T. gondii host cell invasion could be caused by interference with Golgi membrane dynamics. Membrane trafficking molecules are involved in such processes (reviewed in Wei and Seemann, 2010).

Rabs, which are ubiquitously expressed proteins of the RasGTPase superfamily, are important regulators of intracellular membrane traffic. Rabs act by recruiting specific effectors (reviewed in Stenmark, 2009; Liu and Storrie, 2015), and have been described to fulfill important roles in Golgi ribbon organization and trafficking (reviewed in Liu and Storrie, 2012). Rab9A, is of particular interest since it mediates endosome-to trans- Golgi network (TGN) transport (reviewed in Kucera et al., 2016), sorts lysosomal enzymes to late endosomes (Ganley et al., 2004), is involved in lysosome-related organelles biogenesis (Kloer et al., 2010), cation-independent mannose 6-phosphate receptor transport between late endosomes and the TGN (Lombardi et al., 1993), retromer complex dependent transport (Dong et al., 2013), autophagosome formation (Nishida et al., 2009), and Golgi targeting of glycosphingolipids and lipid transport from late endosomes (Choudhury et al., 2002; Walter et al., 2003, 2009; reviewed in Kucera et al., 2016). The role of Rab9A in lipid transport is of particular interest, since both T. gondii and N. caninum have been shown to scavenge cholesterol, ceramides, and sphingolipids from their host cells (Coppens et al., 2006; Romano et al., 2013; Nolan et al., 2015).

In this work, we applied specific antibodies against cis- and trans- Golgi markers (GM130 and TGN46, respectively) and studied the distribution of cis- and trans- Golgi compartments in a retinal pigment epithelial cell line (hTERT-RPE-1) 24 h after infection with B. besnoiti, T. gondii and N. caninum. In addition, we demonstrate the recruitment of Rab9A by these parasites in RPE-1 cells expressing GFP-tagged Rab9A, and an increased expression of Rab9A mRNA and protein in T. gondii infected cells, but not in cells infected with B. besnoiti and N. caninum. In cells expressing Rab9A DN, a dominant negative mutant form of Rab9A, recruitment to the PV upon infection does not take place. Thus, B. besnoiti, T. gondii, and N. caninum differentially modulate the host cell Golgi and endosomal trafficking system.

Section snippets

Chemicals

Biochemical reagents were purchased from Sigma (St. Louis, MO, USA). Culture media and additives were from Thermo Fisher Scientific (former Gibco; Waltham, MA, USA). All primers used in real time PCR were synthesized by Microsynth (Balgach, Switzerland).

Cell culture and parasite culture

Human foreskin fibroblasts (HFF) were grown in Dulbecco's modified Eagle's medium (DMEM), and human telomerase-immortalized retinal pigmented epithelial (hTERT-RPE-1) cells were grown in DMEM/F12. Both media were supplemented with Glutamax, 10%

B. besnoiti, T. gondii, and N. caninum recruit the host Golgi complex to the vicinity of the PV

The Golgi complex of mammalian cells is composed of stacks of flattened cisternae, tubules, and small vesicles. Golgi stacks are divided into three regions: cis-, medial- and trans-. The Golgi stacks associate with two membrane networks located at the cis- and trans-sides, referred to as the cis- Golgi network (CGN - site of entry, close to the endoplasmic reticulum) and the trans- Golgi network (TGN – exit site to distinct cellular locations) (reviewed in Klumperman, 2011). In this study,

Discussion

In this study, the interaction of the Golgi complex of RPE-1 cells infected with B. besnoiti, T. gondii, and N. caninum was comparatively assessed with respect to recruitment to the PV, morphological effects on cis- and trans- Golgi compartments, the recruitment and expression of Rab9A GTPase and the dominant negative mutant form Rab9A DN, and the effects of Rab9A DN overexpression on invasion and replication of these three parasites. Infection with all three parasites resulted in recruitment

Acknowledgements

This work was supported by the Swiss National Science Foundation (grant 310030_165782); and the Swiss Government Excellence Scholarship (reference No. 2015·0062).

We are thankful to Volker Heussler and Mariana De Niz (Institute of Cell Biology, University of Bern) for vectors expressing Rab9A-GFP and Rab9A DN-GFP.

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