Publications

4485

Citrus greening disease, also known as huanglongbing (HLB), is the most devastating disease of Citrus worldwide. This incurable disease is caused primarily by the bacterium Candidatus Liberibacter asiaticus and spread by feeding of the Asian Citrus Psyllid, Diaphorina citri . Ca . L. asiaticus cannot be cultured; its growth is restricted to citrus phloem and the psyllid insect. Management of infected trees includes use of broad-spectrum antibiotics, which have disadvantages. Recent work has sought to identify small molecules that inhibit Ca . L. asiaticus transcription regulators, based on a premise that at least some regulators control expression of genes necessary for virulence. We describe a synthetic, high-throughput screening system to identify compounds that inhibit activity of Ca . L. asiaticus transcription activators LdtR, RpoH, and VisNR. Our system uses the closely related model bacterium, Sinorhizobium meliloti , as a heterologous host for expression of a Ca . L. asiaticus transcription activator, the activity of which is detected through expression of an enhanced green fluorescent protein (EGFP) gene fused to a target promoter. We used this system to screen more than 120,000 compounds for compounds that inhibited regulator activity, but not growth. Our screen identified several dozen compounds that inhibit regulator activity in our assay. This work shows that, in addition to providing a means of characterizing Ca . L. asiaticus regulators, an S. meliloti host can be used for preliminary identification of candidate inhibitory molecules.

Candidatus Liberibacter solanacerum (CLso), transmitted by Bactericera trigonica in a persistent and propagative mode causes carrot yellows disease, inflicting hefty economic losses. Understanding the process of transmission of CLso by psyllids is fundamental to devise sustainable management strategies. Persistent transmission involves critical steps of adhesion, cell invasion, and replication before passage through the midgut barrier. This study uses a transcriptomic approach for the identification of differentially expressed genes with CLso infection in the midguts, adults, and nymphs of B. trigonica and their putative involvement in CLso transmission. Several genes related to focal adhesion and cellular invasion were upregulated after CLso infection. Interestingly, genes involved with proper functionality of the endoplasmic reticulum (ER) were upregulated in CLso infected samples. Notably, genes from the endoplasmic reticulum associated degradation (ERAD) and the unfolded protein response (UPR) pathway were overexpressed after CLso infection. Marker genes of the ERAD and UPR pathways were also upregulated in Diaphorina citri when infected with Candidatus Liberibacter asiaticus (CLas). Upregulation of the ERAD and UPR pathways indicate induction of ER stress by CLso/CLas in their psyllid vector. The role of ER in bacteria–host interactions is well-documented; however, the ER role following pathogenesis of CLso/CLas is unknown and requires further functional validation.

Grapevine (Vitis vinifera L.) is one of the most important horticultural crops in Iran, with >200,000 ha of cultivated area. Recently, outbreaks of the grapevine yellows Bois noir that is associated with phytoplasma strains related to ‘Candidatus Phytoplasma solani’ were recorded in several Iranian regions. This has resulted in severe economic losses. We carried out a survey in 2015, followed by collection of leaf samples from symptomatic grapevines and weeds. Because no information is available on the molecular epidemiology of ‘Ca. P. solani’ in Iran, multiple gene analyses were carried out here according to molecular characterization of the tuf and vmp1 genes. From the molecular characterization, all of the samples (i.e., grapevines, weeds) were infected with tuf b type. Detailed molecular characterization of the vmp1 gene (i.e., PCR–restriction fragment length polymorphism, sequence analysis) defined five molecular types: V1, V4, V10, V15, and V20. The abundance of Convolvulus arvensis in vineyards and detection of the same ‘Ca. P. solani’ molecular types in grapevines and weeds suggest that C. arvensis has a major role in Bois noir epidemiology of Iranian vineyards. Therefore, control strategies should be developed to manage these host plants to reduce inoculum sources of the phytoplasma in vineyards.

Abstract Rickettsiales are a lineage of obligate intracellular Alphaproteobacteria, encompassing important human pathogens, manipulators of host reproduction, and mutualists. Here we report the discovery of a novel Rickettsiales bacterium associated with Paramecium, displaying a unique extracellular lifestyle, including the ability to replicate outside host cells. Genomic analyses show that the bacterium possesses a higher capability to synthesise amino acids, compared to all investigated Rickettsiales. Considering these observations, phylogenetic and phylogenomic reconstructions, and re-evaluating the different means of interaction of Rickettsiales bacteria with eukaryotic cells, we propose an alternative scenario for the evolution of intracellularity in Rickettsiales. According to our reconstruction, the Rickettsiales ancestor would have been an extracellular and metabolically versatile bacterium, while obligate intracellularity would have evolved later, in parallel and independently, in different sub-lineages. The proposed new scenario could impact on the open debate on the lifestyle of the last common ancestor of mitochondria within Alphaproteobacteria.

Abstract The actinobacterial acI lineage is among the most successful and ubiquitous freshwater bacterioplankton found on all continents, often representing more than half of all microbial cells in the lacustrine environment and constituting multiple ecotypes. However, stably growing pure cultures of the acI lineage have not been established despite various cultivation efforts based on ecological and genomic studies on the lineage, which is in contrast to the ocean from which abundant microorganisms such as Prochlorococcus, Pelagibacter, and Nitrosopumilus have been isolated. Here, we report the first two pure cultures of the acI lineage successfully maintained by supplementing the growth media with catalase. Catalase was critical for stabilizing the growth of acI strains irrespective of the genomic presence of the catalase-peroxidase (katG) gene. The two strains, representing two novel species, displayed differential phenotypes and distinct preferences for reduced sulfurs and carbohydrates, some of which were difficult to predict based on genomic information. Our results suggest that culture of previously uncultured freshwater bacteria can be facilitated by a simple catalase-supplement method and indicate that genome-based metabolic prediction can be complemented by physiological analyses.

Abstract Human activities such as deforestation, urbanization, and environmental pollution lead to a reduction in the spatial boundary between wild animals, domestic animals and humans. These activities increase the risk for the emergence of pathogens from the sylvatic cycle in the population of domestic animals and humans. Foxes are recognized as potential reservoirs for a number of bacterial pathogens of medical and public health concern. The aim of the present study was to investigate the prevalence and spatial distribution of bacterial tick-borne pathogens from the Anaplasmataceae family, Borrelia burgdorferi sensu lato (s.l.), Rickettsia spp., Coxiella burnetii, Francisella tularensis, Bartonella spp., in the red fox population from Serbia and to discuss the obtained results from the epidemiological point of view. Legally hunted red foxes (Vulpes vulpes) from 14 localities in Serbia were included in the study and spleen samples from 129 animals were tested with conventional PCR assays for the presence of bacterial tick-borne pathogens. DNA of Candidatus Neoehrlichia sp. (FU98), Borrelia burgdorferi sensu stricto, Borrelia lusitaniae, and Borrelia garinii was detected in 6 (4.7%), 1 (0.8%), 2 (1.6%) and 1 (0.8%) animals, respectively. Co-infection by Candidatus Neoehrlichia sp. (FU98) and B. garinii was detected in one animal. All samples were negative for other tested bacterial tick-borne pathogens. The results of the present study indicate the potential role of foxes in natural cycles of Candidatus Neoehrlichia sp. (FU98) and causative agents of Lyme borreliosis in the investigated areas. Further research is required to elucidate the role of foxes in the epidemiology of these and other tick-borne zoonotic pathogens in the Republic of Serbia.

Citrus huanglongbing (HLB) is one of the most destructive diseases affecting citrus plants. ‘Candidatus Liberibacter asiaticus’, an uncultivated α-proteobacteria, is the most widely spread causal agent of HLB and is transmitted by the Asian citrus psyllid Diaphorina citri. ‘Ca. L. asiaticus’ attachment to the psyllid midgut is believed to be critical to further infect other organs, including the salivary gland. In this study, the type IVc tight adherence (Tad) pilus locus encoded by ‘Ca. L. asiaticus’ was characterized. The Tad loci are conserved among members of Rhizobiaceae, including ‘Ca. L. asiaticus’ and Agrobacterium spp. Ectopic expression of the ‘Ca. L. asiaticus’ cpaF gene, an ATPase essential for the biogenesis and secretion of the Tad pilus, restored the adherence phenotype in cpaF mutant of A. tumefaciens, indicating CpaF of ‘Ca. L. asiaticus’ was functional and critical for bacterial adherence mediated by Tad pilus. Quantitative reverse transcription PCR (qRT-PCR) analysis revealed that ‘Ca. L. asiaticus’ Tad pilus-encoding genes and ‘Ca. L. asiaticus’ pilin gene flp3 were upregulated in psyllids compared with in planta. A bacterial one-hybrid assay showed that ‘Ca. L. asiaticus’ VisN and VisR, members of the LuxR transcriptional factor family, were bound to the flp3 promoter. VisNR regulate flp3. Negative regulation of the flp3 promoter by both VisN and VisR was demonstrated using a shuttle strategy, with analysis of the phenotypes and immunoblotting together with quantification of the expression of the flp3 promoter fused to the β-galactosidase reporter gene. Comparative expression analysis confirmed that ‘Ca. L. asiaticus’ visNR was less expressed in the psyllid than in the plant host. Further, motility and biofilm phenotypes of the visNR mutant of A. tumefaciens were fully complemented by expressing ‘Ca. L. asiaticus’ visNR together. The physical interaction between VisN and VisR was confirmed by pull-down and stability assays. The interaction of the flp3 promoter with VisR was verified by electrophoretic mobility shift assay. Taken together, the results revealed the contribution of the Tad pilus apparatus in the colonization of the insect vector by ‘Ca. L. asiaticus’ and shed light on the involvement of VisNR in regulation of the Tad locus.