Candida albicans is able to use M cells as a portal of entry across the intestinal barrier in vitro.

Candida albicans is the most frequent yeast responsible for systemic infections in humans. These infections mainly originate from the gastrointestinal tract where C. albicans can invade the gut epithelial barrier to gain access to the bloodstream. Along the gut, pathogens can use Microfold (M) cells as a portal of entry to cross the epithelial barrier. M cells are specialized cells mainly located in the follicule-associated epithelium of Peyer patches. In this study, we used scanning electron and fluorescence microscopy, adhesion and invasion assays and fungal mutants to investigate the interactions of C. albicans with M cells obtained in an established in vitro model whereby enterocyte-like Caco-2 cells co-cultured with the Raji B cell line undergo a phenotypic switch to morphologically and functionally resembling M cells. Our data demonstrate that C. albicans co-localizes with and invades preferentially M cells, providing evidence that the fungus can use M cells as a portal of entry into the intestinal barrier. In addition to active penetration, F-actin dependent endocytosis contributes to internalization of the fungus into M cells through a mechanism involving hypha-associated invasins including Ssa1 and Als3.

The origin of intraspecific variation of virulence in an eukaryotic immune suppressive parasite.

Occurrence of intraspecific variation in parasite virulence, a prerequisite for coevolution of hosts and parasites, has largely been reported. However, surprisingly little is known of the molecular bases of this variation in eukaryotic parasites, with the exception of the antigenic variation used by immune-evading parasites of mammals. The present work aims to address this question in immune suppressive eukaryotic parasites. In Leptopilina boulardi, a parasitic wasp of Drosophila melanogaster, well-defined virulent and avirulent strains have been characterized. The success of virulent females is due to a major immune suppressive factor, LbGAP, a RacGAP protein present in the venom and injected into the host at oviposition. Here, we show that an homologous protein, named LbGAPy, is present in the venom of the avirulent strain. We then question whether the difference in virulence between strains originates from qualitative or quantitative differences in LbGAP and LbGAPy proteins. Results show that the recombinant LbGAPy protein has an in vitro GAP activity equivalent to that of recombinant LbGAP and similarly targets Drosophila Rac1 and Rac2 GTPases. In contrast, a much higher level of both mRNA and protein is found in venom-producing tissues of virulent parasitoids. The F1 offspring between virulent and avirulent strains show an intermediate level of LbGAP in their venom but a full success of parasitism. Interestingly, they express almost exclusively the virulent LbGAP allele in venom-producing tissues. Altogether, our results demonstrate that the major virulence factor in the wasp L. boulardi differs only quantitatively between virulent and avirulent strains, and suggest the existence of a threshold effect of this molecule on parasitoid virulence. We propose that regulation of gene expression might be a major mechanism at the origin of intraspecific variation of virulence in immune suppressive eukaryotic parasites. Understanding this variation would improve our knowledge of the mechanisms of transcriptional evolution currently under active investigation.

Hosting certain facultative symbionts modulates the phenoloxidase activity and immune response of the pea aphid Acyrthosiphon pisum.

The pea aphid Acyrthosiphon pisum hosts different facultative symbionts (FS) which provide it with various benefits, such as tolerance to heat or protection against natural enemies (e.g., fungi, parasitoid wasps). Here, we investigated whether and how the presence of certain FS could affect phenoloxidase (PO) activity, a key component of insect innate immunity, under normal and stressed conditions. For this, we used clones of A. pisum of different genetic backgrounds (LL01, YR2 and T3-8V1) lacking FS or harboring one or two (Regiella insecticola, Hamiltonella defensa, Serratia symbiotica + Rickettsiella viridis). Gene expression and proteomics analyses of the aphid hemolymph indicated that the two A. pisum POs, PPO1 and PPO2, are expressed and translated into proteins. The level of PPO genes expression as well as the amount of PPO proteins and phenoloxidase activity in the hemolymph depended on both the aphid genotype and FS species. In particular, H. defensa and R. insecticola, but not S. symbiotica + R. viridis, caused a sharp decrease in PO activity by interfering with both transcription and translation. The microinjection of different types of stressors (yeast, Escherichia coli, latex beads) in the YR2 lines hosting different symbionts affected the survival rate of aphids and, in most cases, also decreased the expression of PPO genes after 24 h. The amount and activity of PPO proteins varied according to the type of FS and stressor, without clear corresponding changes in gene expression. These data demonstrate that the presence of certain FS influences an important component of pea aphid immunity.

Convergent use of RhoGAP toxins by eukaryotic parasites and bacterial pathogens.

Inactivation of host Rho GTPases is a widespread strategy employed by bacterial pathogens to manipulate mammalian cellular functions and avoid immune defenses. Some bacterial toxins mimic eukaryotic Rho GTPase-activating proteins (GAPs) to inactivate mammalian GTPases, probably as a result of evolutionary convergence. An intriguing question remains whether eukaryotic pathogens or parasites may use endogenous GAPs as immune-suppressive toxins to target the same key genes as bacterial pathogens. Interestingly, a RhoGAP domain-containing protein, LbGAP, was recently characterized from the parasitoid wasp Leptopilina boulardi, and shown to protect parasitoid eggs from the immune response of Drosophila host larvae. We demonstrate here that LbGAP has structural characteristics of eukaryotic RhoGAPs but that it acts similarly to bacterial RhoGAP toxins in mammals. First, we show by immunocytochemistry that LbGAP enters Drosophila immune cells, plasmatocytes and lamellocytes, and that morphological changes in lamellocytes are correlated with the quantity of LbGAP they contain. Demonstration that LbGAP displays a GAP activity and specifically interacts with the active, GTP-bound form of the two Drosophila Rho GTPases Rac1 and Rac2, both required for successful encapsulation of Leptopilina eggs, was then achieved using biochemical tests, yeast two-hybrid analysis, and GST pull-down assays. In addition, we show that the overall structure of LbGAP is similar to that of eukaryotic RhoGAP domains, and we identify distinct residues involved in its interaction with Rac GTPases. Altogether, these results show that eukaryotic parasites can use endogenous RhoGAPs as virulence factors and that despite their differences in sequence and structure, eukaryotic and bacterial RhoGAP toxins are similarly used to target the same immune pathways in insects and mammals.

Enterocyte Purge and Rapid Recovery Is a Resilience Reaction of the Gut Epithelium to Pore-Forming Toxin Attack.

Besides digesting nutrients, the gut protects the host against invasion by pathogens. Enterocytes may be subjected to damage by both microbial and host defensive responses, causing their death. Here, we report a rapid epithelial response that alleviates infection stress and protects the enterocytes from the action of microbial virulence factors. Intestinal epithelia exposed to hemolysin, a pore-forming toxin secreted by Serratia marcescens, undergo an evolutionarily conserved process of thinning followed by the recovery of their initial thickness within a few hours. In response to hemolysin attack, Drosophila melanogaster enterocytes extrude most of their apical cytoplasm, including damaged organelles such as mitochondria, yet do not lyse. We identify two secreted peptides, the expression of which requires CyclinJ, that mediate the recovery phase in which enterocytes regain their original shape and volume. Epithelial thinning and recovery constitute a fast and efficient response to intestinal infections, with pore-forming toxins acting as alarm signals.

The cellular immune response of the pea aphid to foreign intrusion and symbiotic challenge.

Recent studies suggest that the pea aphid (Acyrthosiphon pisum) has low immune defenses. However, its immune components are largely undescribed, and notably, extensive characterization of circulating cells has been missing. Here, we report characterization of five cell categories in hemolymph of adults of the LL01 pea aphid clone, devoid of secondary symbionts (SS): prohemocytes, plasmatocytes, granulocytes, spherulocytes and wax cells. Circulating lipid-filed wax cells are rare; they otherwise localize at the basis of the cornicles. Spherulocytes, that are likely sub-cuticular sessile cells, are involved in the coagulation process. Prohemocytes have features of precursor cells. Plasmatocytes and granulocytes, the only adherent cells, can form a layer in vivo around inserted foreign objects and phagocytize latex beads or Escherichia coli bacteria injected into aphid hemolymph. Using digital image analysis, we estimated that the hemolymph from one LL01 aphid contains about 600 adherent cells, 35% being granulocytes. Among aphid YR2 lines differing only in their SS content, similar results to LL01 were observed for YR2-Amp (without SS) and YR2-Ss (with Serratia symbiotica), while YR2-Hd (with Hamiltonella defensa) and YR2(Ri) (with Regiella insecticola) had strikingly lower adherent hemocyte numbers and granulocyte proportions. The effect of the presence of SS on A. pisum cellular immunity is thus symbiont-dependent. Interestingly, Buchnera aphidicola (the aphid primary symbiont) and all SS, whether naturally present, released during hemolymph collection, or artificially injected, were internalized by adherent hemocytes. Inside hemocytes, SS were observed in phagocytic vesicles, most often in phagolysosomes. Our results thus raise the question whether aphid symbionts in hemolymph are taken up and destroyed by hemocytes, or actively promote their own internalization, for instance as a way of being transmitted to the next generation. Altogether, we demonstrate here a strong interaction between aphid symbionts and immune cells, depending upon the symbiont, highlighting the link between immunity and symbiosis.