Marie-Pierre Dubrana

Invasion of insect cells by Spiroplasma citri involves spiralin relocalization and lectin/glycoconjugate-type interactions.

Spiroplamas are helical, cell wall‐less bacteria belonging to the Class Mollicutes, a group of microorganisms phylogenetically related to low G+C, Gram‐positive bacteria. Spiroplasma species are all found associated with arthropods and a few, including Spiroplasma citri are pathogenic to plant. Thus S. citri has the ability to colonize cells of two very distinct hosts, the plant and the insect vector. While spiroplasmal factors involved in transmission by the leafhopper Circulifer haematoceps have been identified, their specific contribution to invasion of insect cells is poorly understood. In this study we provide evidence that the lipoprotein spiralin plays a major role in the very early step of cell invasion. Confocal laser scanning immunomicroscopy revealed a relocalization of spiralin at the contact zone of adhering spiroplasmas. The implication of a role for spiralin in adhesion to insect cells was further supported by adhesion assays showing that a spiralin‐less mutant was impaired in adhesion and that recombinant spiralin triggered adhesion of latex beads. We also showed that cytochalasin D induced changes in the surface‐exposed glycoconjugates, as inferred from the lectin binding patterns, and specifically improved adhesion of S. citri wild‐type but not of the spiralin‐less mutant. These results indicate that cytochalasin D exposes insect cell receptors of spiralin that are masked in untreated cells. In addition, competitive adhesion assays with lectins strongly suggest spiralin to exhibit glycoconjugate binding properties similar to that of the Vicia villosa agglutinin (VVA) lectin.

The Repetitive Domain of ScARP3d Triggers Entry of Spiroplasma citri into Cultured Cells of the Vector Circulifer haematoceps

Spiroplasma citri is a plant pathogenic mollicute transmitted by the leafhopper vector Circulifer haematoceps. Successful transmission requires the spiroplasmas to cross the intestinal epithelium and salivary gland barriers through endocytosis mediated by receptor-ligand interactions. To characterize these interactions we studied the adhesion and invasion capabilities of a S. citri mutant using the Ciha-1 leafhopper cell line. S. citri GII3 wild-type contains 7 plasmids, 5 of which (pSci1 to 5) encode 8 related adhesins (ScARPs). As compared to the wild-type strain GII3, the S. citri mutant G/6 lacking pSci1 to 5 was affected in its ability to adhere and enter into the Ciha-1 cells. Proteolysis analyses, Triton X-114 partitioning and agglutination assays showed that the N-terminal part of ScARP3d, consisting of repeated sequences, was exposed to the spiroplasma surface whereas the C-terminal part was anchored into the membrane. Latex beads cytadherence assays showed the ScARP3d repeat domain (Rep3d) to be involved, and internalization of the Rep3d-coated beads to be actin-dependent. These data suggested that ScARP3d, via its Rep3d domain, was implicated in adhesion of S. citri GII3 to insect cells. Inhibition tests using anti-Rep3d antibodies and competitive assays with recombinant Rep3d both resulted in a decrease of insect cells invasion by the spiroplasmas. Unexpectedly, treatment of Ciha-1 cells with the actin polymerisation inhibitor cytochalasin D increased adhesion and consequently entry of S. citri GII3. For the ScARPs-less mutant G/6, only adhesion was enhanced though to a lesser extent following cytochalasin D treatment. All together these results strongly suggest a role of ScARPs, and particularly ScARP3d, in adhesion and invasion of the leafhopper cells by S. citri.

Entry of Spiroplasma citri into Circulifer haematoceps Cells Involves Interaction between Spiroplasma Phosphoglycerate Kinase and Leafhopper Actin

ABSTRACT Transmission of the phytopathogenic mollicutes, spiroplasmas, and phytoplasmas by their insect vectors mainly depends on their ability to pass through gut cells, to multiply in various tissues, and to traverse the salivary gland cells. The passage of these different barriers suggests molecular interactions between the plant mollicute and the insect vector that regulate transmission. In the present study, we focused on the interaction between Spiroplasma citri and its leafhopper vector, Circulifer haematoceps. An in vitro protein overlay assay identified five significant binding activities between S. citri proteins and insect host proteins from salivary glands. One insect protein involved in one binding activity was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as actin. Confocal microscopy observations of infected salivary glands revealed that spiroplasmas colocated with the host actin filaments. An S. citri actin-binding protein of 44 kDa was isolated by affinity chromatography and identified by LC-MS/MS as phosphoglycerate kinase (PGK). To investigate the role of the PGK-actin interaction, we performed competitive binding and internalization assays on leafhopper cultured cell lines (Ciha-1) in which His6-tagged PGK from S. citri or purified PGK from Saccharomyces cerevisiae was added prior to the addition of S. citri inoculum. The results suggested that exogenous PGK has no effect on spiroplasmal attachment to leafhopper cell surfaces but inhibits S. citri internalization, demonstrating that the process leading to internalization of S. citri in eukaryotic cells requires the presence of PGK. PGK, regardless of origin, reduced the entry of spiroplasmas into Ciha-1 cells in a dose-dependent manner.

Sequences Essential for Transmission of Spiroplasma citri by Its Leafhopper Vector, Circulifer haematoceps, Revealed by Plasmid Curing and Replacement Based on Incompatibility†

ABSTRACT Spiroplasma citri GII3 contains highly related low-copy-number plasmids pSci1 to -6. Despite the strong similarities between their replication regions, these plasmids coexist in the spiroplasma cells, indicating that they are mutually compatible. The pSci1 to -6 plasmids encode the membrane proteins known as S. citri adhesion-related proteins (ScARPs) (pSci1 to -5) and the hydrophilic protein P32 (pSci6), which had been tentatively associated with insect transmission, as they were not detected in non-insect-transmissible strains. With the aim of further investigating the role of plasmid-encoded determinants in insect transmission, we have constructed S. citri mutant strains that differ in their plasmid contents by developing a plasmid curing/replacement strategy based on the incompatibility of plasmids having identical replication regions. Experimental transmission of these S. citri plasmid mutants through injection into the leafhopper vector Circulifer haematoceps revealed that pSci6, more precisely, the pSci6_06 coding sequence, encoding a protein of unknown function, was essential for transmission. In contrast, ScARPs and P32 were dispensable for both acquisition and transmission of the spiroplasmas by the leafhopper vector, even though S. citri mutants lacking pSci1 to -5 (encoding ScARPs) were acquired and transmitted at lower efficiencies than the wild-type strain GII3.

Involvement of a Minimal Actin-Binding Region of Spiroplasma citri Phosphoglycerate Kinase in Spiroplasma Transmission by Its Leafhopper Vector

Background Spiroplasma citri is a wall-less bacterium that colonizes phloem vessels of a large number of host plants. Leafhopper vectors transmit S. citri in a propagative and circulative manner, involving colonization and multiplication of bacteria in various insect organs. Previously we reported that phosphoglycerate kinase (PGK), the well-known glycolytic enzyme, bound to leafhopper actin and was unexpectedly implicated in the internalization process of S. citri into Circulifer haematoceps cells. Methodology/Principal Findings In an attempt to identify the actin-interacting regions of PGK, several overlapping PGK truncations were generated. Binding assays, using the truncations as probes on insect protein blots, revealed that the actin-binding region of PGK was located on the truncated peptide designated PGK-FL5 containing amino acids 49–154. To investigate the role of PGK-FL5-actin interaction, competitive spiroplasma attachment and internalization assays, in which His6-tagged PGK-FL5 was added to Ciha-1 cells prior to infection with S. citri, were performed. No effect on the efficiency of attachment of S. citri to leafhopper cells was observed while internalization was drastically reduced. The in vivo effect of PGK-FL5 was confirmed by competitive experimental transmission assays as injection of PGK-FL5 into S. citri infected leafhoppers significantly affected spiroplasmal transmission. Conclusion These results suggest that S. citri transmission by its insect vector is correlated to PGK ability to bind actin.

Variable membrane protein A of flavescence dorée phytoplasma binds the midgut perimicrovillar membrane of Euscelidius variegatus and promotes adhesion to its epithelial cells

ABSTRACT Phytoplasmas are uncultivated plant pathogens and cell wall-less bacteria and are transmitted from plant to plant by hemipteran insects. The phytoplasmas circulative propagative cycle in insects requires the crossing of the midgut and salivary glands, and primary adhesion to cells is an initial step toward the invasion process. The flavescence dorée (FD) phytoplasma possesses a set of variable membrane proteins (Vmps) exposed on its surface, and this pathogen is suspected to interact with insect cells. The results showed that VmpA is expressed by the flavescence dorée phytoplasma present in the midgut and salivary glands. Phytoplasmas cannot be cultivated at present, and no mutant can be produced to investigate the putative role of Vmps in the adhesion of phytoplasma to insect cells. To overcome this difficulty, we engineered the Spiroplasma citri mutant G/6, which lacks the ScARP adhesins, for VmpA expression and used VmpA-coated fluorescent beads to determine if VmpA acts as an adhesin in ex vivo adhesion assays and in vivo ingestion assays. VmpA specifically interacted with Euscelidius variegatus insect cells in culture and promoted the retention of VmpA-coated beads to the midgut of E. variegatus. In this latest case, VmpA-coated fluorescent beads were localized and embedded in the perimicrovillar membrane of the insect midgut. Thus, VmpA functions as an adhesin that could be essential in the colonization of the insect by the FD phytoplasmas. IMPORTANCE Phytoplasmas infect a wide variety of plants, ranging from wild plants to cultivated species, and are transmitted by different leafhoppers, planthoppers, and psyllids. The specificity of the phytoplasma-insect vector interaction has a major impact on the phytoplasma plant host range. As entry into insect cells is an obligate process for phytoplasma transmission, the bacterial adhesion to insect cells is a key step. Thus, studying surface-exposed proteins of phytoplasma will help to identify the adhesins implicated in the specific recognition of insect vectors. In this study, it is shown that the membrane protein VmpA of the flavescence dorée (FD) phytoplasma acts as an adhesin that is able to interact with cells of Euscelidius variegatus, the experimental vector of the FD phytoplasma.

Differential expression of Spiroplasma citri surface protein genes in the plant and insect hosts.

BackgroundSpiroplasma citri is a cell wall-less, plant pathogenic bacteria that colonizes two distinct hosts, the leafhopper vector and the host plant. Given the absence of a cell wall, surface proteins including lipoproteins and transmembrane polypeptides are expected to play key roles in spiroplasma/host interactions. Important functions in spiroplasma/insect interactions have been shown for a few surface proteins such as the major lipoprotein spiralin, the transmembrane S. citri adhesion-related proteins (ScARPs) and the sugar transporter subunit Sc76. S. citri efficient transmission from the insect to the plant is expected to rely on its ability to adapt to the different environments and more specifically to regulate the expression of genes encoding surface-exposed proteins.ResultsGenes encoding S. citri lipoproteins and ScARPs were investigated for their expression level in axenic medium, in the leafhopper vector Circulifer haematoceps and in the host plant (periwinkle Catharanthus roseus) either insect-infected or graft-inoculated. The vast majority of the lipoprotein genes tested (25/28) differentially responded to the various host environments. Considering their relative expression levels in the different environments, the possible involvement of the targeted genes in spiroplasma host adaptation was discussed. In addition, two S. citri strains differing notably in their ability to express adhesin ScARP2b and pyruvate dehydrogenase E1 component differed in their capacity to multiply in the two hosts, the plant and the leafhopper vector.ConclusionsThis study provided us with a list of genes differentially expressed in the different hosts, leading to the identification of factors that are thought to be involved in the process of S. citri host adaptation. The identification of such factors is a key step for further understanding of S. citri pathogenesis. Moreover the present work highlights the high capacity of S. citri in tightly regulating the expression level of a large set of surface protein genes, despite the small size of its genome.

Two Phytoplasmas Elicit Different Responses in the Insect Vector Euscelidius variegatus Kirschbaum

ABSTRACT Phytoplasmas are plant-pathogenic bacteria transmitted by hemipteran insects. The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma (CYp) and a laboratory vector of flavescence dorée phytoplasma (FDp). The two phytoplasmas induce different effects on this species: CYp slightly improves whereas FDp negatively affects insect fitness. To investigate the molecular bases of these different responses, transcriptome sequencing (RNA-seq) analysis of E. variegatus infected with either CYp or FDp was performed. The sequencing provided the first de novo transcriptome assembly for a phytoplasma vector and a starting point for further analyses on differentially regulated genes, mainly related to immune system and energy metabolism. Insect phenoloxidase activity, immunocompetence, and body pigmentation were measured to investigate the immune response, while respiration and movement rates were quantified to confirm the effects on energy metabolism. The activation of the insect immune response upon infection with FDp, which is not naturally transmitted by E. variegatus, confirmed that this bacterium is mostly perceived as a potential pathogen. Conversely, the acquisition of CYp, which is naturally transmitted by E. variegatus, seems to increase the insect fitness by inducing a prompt response to stress. This long-term relationship is likely to improve survival and dispersal of the infected insect, thus enhancing the opportunity of phytoplasma transmission.

Proteolytic Post-Translational Processing of Adhesins in a Pathogenic Bacterium

Mollicutes, including mycoplasmas and spiroplasmas, have been considered as good representatives of the « minimal cell » concept: these wall-less bacteria are small in size and possess a minimal genome and restricted metabolic capacities. However, the recent discovery of the presence of post-translational modifications unknown so far, such as the targeted processing of membrane proteins of mycoplasma pathogens for human and swine, revealed a part of the hidden complexity of these microorganisms. In this study, we show that in the phytopathogen, insect-vectored Spiroplasma citri GII-3 adhesion-related protein (ScARP) adhesins are post-translationally processed through an ATP-dependent targeted cleavage. The cleavage efficiency could be enhanced in vitro when decreasing the extracellular pH or upon the addition of polyclonal antibodies directed against ScARP repeated units, suggesting that modification of the surface charge and/or ScARP conformational changes could initiate the cleavage. The two major sites for primary cleavage are localized within predicted disordered regions and do not fit any previously reported cleavage motif; in addition, the inhibition profile and the metal ion requirements indicate that this post-translational modification involves at least one non-conventional protease. Such a proteolytic process may play a role in S. citri colonization of cells of the host insect. Furthermore, our work indicates that post-translational cleavage of adhesins represents a common feature to mollicutes colonizing distinct hosts and that processing of surface antigens could represent a way to make the most out of a minimal genome.

Cryptic haplotypes of "Candidatus Liberibacter africanus".

“Candidatus Liberibacter africanus” (Laf) has long been recognised as a causal agent of the devastating citrus disease huanglongbing (HLB) or citrus greening. This species is currently restricted to Africa, the Arabian Peninsula and some Indian Ocean islands and is vectored by the African citrus psyllid, Trioza erytreae. Blotchy mottle on citrus leaves is characteristic of the disease. Somewhat similar symptoms in the rutaceous tree Calodendrum capensis (Cape chestnut) resulted in the discovery of Laf outside commercial citrus crops in South Africa. This was classed as a subspecies of Laf (capensis, hence LafC). In subsequent surveys of commercial citrus crops and Calodendrum, both natural and ornamental specimens, LafC was not found in citrus, nor has Laf been found in C. capensis. HLB was reported from Madagascar in 1968 but no sequences from this source have so far been published. Until fairly recently, only the reference 16S rRNA gene sequences of Laf (L22533) and LafC (AF137368) had been deposited in GenBank. Both of these reference sequences contain a number of unresolved nucleotides. When these nucleotide positions are invstigated by aligning against more recently available sequences, it becomes evident that these unresolved positions represent one percentage point difference in similarity between Laf and LafC. The originally reported 97.4% similarity is therefore incorrect based on this new information. Recalculating the similarity on the full length 16S rDNA sequence results in 99.54% similarity, a value too high to justify a subspecies status. LafC should therefore be downgraded as a haplotype of Laf. Further, the six 16S rRNA gene sequences currently available in GenBank identified as Laf as a species separate into two haplotype groups. The three haplotypes of Laf are therefore: (i) LafA, designated as the first accession sequenced (L22533); (ii) LafC, i.e the former subspecies “capensis”, to recognise the priority in the use of this term; (iii) LafB, the third previously unrecognised haplotype. Thus the cryptic presence of three haplotypes is revealed by this review of the Laf 16S rDNA sequences.