Publications

4372

Summary ‘ Candidatus Liberibacter asiaticus’ (CLas) is a phloem‐limited non‐culturable α‐proteobacterium associated with citrus Huanglongbing, a highly destructive disease threatening global citrus industry. Research on CLas is challenging due to the current inability to culture CLas in vitro and the low CLas titre in citrus plant. Here, we develop a CLas enrichment system using the holoparasitic dodder plant ( Cuscuta campestris ) as an amenable host to acquire and enrich CLas from CLas‐infected citrus shoots maintained hydroponically. Forty‐eight out of fifty‐five (87%) dodder plants successfully parasitized CLas‐infected citrus shoots with detectable CLas by PCR. Among 48 dodders cultures, 30 showed two‐ to 419‐fold CLas titre increase as compared to the corresponding citrus hosts. The CLas population rapidly increased and reached the highest level in dodder tendrils at 15 days after parasitizing citrus shoot. Genome sequencing and assembly derived from CLas‐enriched dodder DNA samples generated a higher resolution than those obtained for CLas from citrus hosts. No genomic variation was detected in CLas after transmission from citrus to dodder during short‐term parasitism. Dual RNA‐Seq experiments showed similar CLas gene expression profiles in dodder and citrus samples, yet dodder samples generated a higher resolution of CLas transcriptome data. The ability of dodder to support CLas multiplication to high levels, as well as its advantage in CLas genomic and transcriptomic analyses, make it an optimal model for further studies on CLas–host interaction.

Summary Members of the bacterial order Rickettsiales are obligatorily associated with a wide range of eukaryotic hosts. Their evolutionary trajectories, in particular concerning the origin of shared or differential traits among distant sub‐lineages, are still poorly understood. Here, we characterized a novel Rickettsiales bacterium associated with the ciliate Paramecium tredecaurelia and phylogenetically related to the Rickettsia genus. Its genome encodes significant lineage‐specific features, chiefly the mevalonate pathway gene repertoire, involved in isoprenoid precursor biosynthesis. Not only this pathway has never been described in Rickettsiales , it also is very rare among bacteria, though typical in eukaryotes, thus likely representing a horizontally acquired trait. The presence of these genes could enable an efficient exploitation of host‐derived intermediates for isoprenoid synthesis. Moreover, we hypothesize the reversed reactions could have replaced canonical pathways for producing acetyl‐CoA, essential for phospholipid biosynthesis. Additionally, we detected phylogenetically unrelated mevalonate pathway genes in metagenome‐derived Rickettsiales sequences, likely indicating evolutionary convergent effects of independent horizontal gene transfer events. Accordingly, convergence, involving both gene acquisitions and losses, is highlighted as a relevant evolutionary phenomenon in Rickettsiales , possibly favoured by plasticity and comparable lifestyles, representing a potentially hidden origin of other more nuanced similarities among sub‐lineages.

The Asian citrus psyllid Diaphorina citri is the transmission vector of Huanglongbing (HLB), a devastating disease of citrus plants. The bacterium “Candidatus Liberibacter asiaticus” (CLas) associated with HLB is transmitted between host plants by D. citri in a circulative manner. Understanding the interaction between CLas and its insect vector is key for protecting citrus cultivation from HLB damage. Here, we used RNA sequencing and liquid chromatography-mass spectrometry (LC-MS) to analyze the transcriptome and metabolome of D. citri interacting with CLas. We identified 662 upregulated and 532 downregulated genes in CLas-infected insects. These genes were enriched in pathways involving carbohydrate metabolism, the insects’ immune system, and metabolism of cofactors and vitamins. We also detected 105 differential metabolites between CLas-infected and non-infected insects, including multiple nucleosides and lipid-related molecules. The integrated analysis revealed nine pathways—including those of the glycine, serine, threonine, and purine metabolism—affected by the differentially expressed genes from both groups. The network for these pathways was subsequently constructed. Our results thus provide insights regarding the cross-talk between the transcriptomic and metabolomic changes in D. citri in response to CLas infection, as well as information on the pathways and genes/metabolites related to the CLas–D. citri interaction.

Abstract Background Hemotropic mycoplasmas (hemoplasmas) infect animals and humans and can lead to clinical syndromes mainly characterized by hemolytic anemia. A novel pathogen, Candidatus Mycoplasma haemohominis, was recently associated with a case of human hemoplasmosis in Europe. Here we report the first detection of this pathogen in an Australian patient exhibiting persistent fever, hemolytic anemia, and pancytopenia over a 10-month period. Methods After exhaustive negative testing for human infectious diseases, whole genome sequencing (WGS) was performed on the patient’s bone marrow aspirate, using an Illumina NextSeq500 platform. Conventional polymerase chain reaction (PCR), followed by Sanger sequencing, was then performed on blood samples using novel Mycoplasma-specific primers targeting the 16S ribosomal RNA gene. In addition, a Mycoplasma-specific fluorescence in situ hybridization (FISH) assay was developed to differentiate Mycoplasma cells from other erythrocyte inclusions (eg, Pappenheimer and Howell-Jolly bodies) which are morphologically similar to bacterial cocci by light microscopy. Results WGS analysis revealed that approximately 0.04% of the total number of unmapped reads to human genome corresponded to Mycoplasma species. A 1-kb Mycoplasma 16S fragment was successfully amplified by conventional PCR, and sequence analyses revealed 100% identity with Candidatus Mycoplasma haemohominis. FISH confirmed that several (approximately 2%) epierythrocytic inclusions initially observed by light microscopy corresponded to Mycoplasma cells. Conclusions This represents the second report of hemolytic anemia associated with hemoplasma infection in a human, and the first report of human hemoplasmosis in Australia. This study highlights the importance of new and emerging diagnostic approaches and need for further investigations on the epidemiology of Candidatus Mycoplasma haemohominis in Australia.