Biochemistry

Rickettsiales (Rickettsia spp., Ehrlichia spp., and Anaplasma spp., etc.) are generally recognized as potentially emerging tick-borne pathogens. However, some bacteria and areas in China remain uninvestigated. In this study, we collected 113 ticks from mammals in Guizhou Province, Southwest China, and screened for the Rickettsiales bacteria. Subsequently, two spotted fever group Rickettsia species and one Candidatus Lariskella sp. were detected and characterized. “Candidatus Rickettsia jingxinensis” was detected in Rhipicephalus microplus (1/1), Haemaphysalis flava (1/3, 33.33%), Haemaphysalis kitaokai (1/3), and Ixodes sinensis (4/101, 3.96%), whereas Rickettsia monacensis was positive in H. flava (1/3), H. kitaokai (2/3), and I. sinensis ticks (74/101, 73.27%). At least two variants/sub-genotypes were identified in the R. monacensis isolates, and the strikingly high prevalence of R. monacensis may suggest a risk of human infection. Unexpectedly, a Candidatus Lariskella sp. belonging to the family Candidatus Midichloriaceae was detected from Ixodes ovatus (1/4) and I. sinensis (10/101, 9.90%). The gltA and groEL gene sequences were successfully obtained, and they show the highest (74.63–74.89% and 73.31%) similarities to “Candidatus Midichloria mitochondrii”, respectively. Herein, we name the species “Candidatus Lariskella guizhouensis”. These may be the first recovered gltA and groEL sequences of the genus Candidatus Lariskella.

AbstractCandidatus Branchiomonas cysticola is an intracellular, gram-negative Betaproteobacteria causing epitheliocystis in Atlantic Salmon (Salmo salar L.). The bacterium has not been genetically characterized at the intraspecific level despite its high prevalence among salmon suffering from gill disease in Norwegian aquaculture. DNA from gill samples of Atlantic salmon PCR positive for Cand. B. cysticola and displaying pathological signs of gill disease, was, therefore, extracted and subject to next-generation sequencing (mNGS). Partial sequences of four housekeeping (HK) genes (aceE, lepA, rplB, rpoC) were ultimately identified from the sequenced material. Assays for real-time RT-PCR and fluorescence in-situ hybridization, targeting the newly acquired genes, were simultaneously applied with existing assays targeting the previously characterized 16S rRNA gene. Agreement in both expression and specificity between these putative HK genes and the 16S gene was observed in all instances, indicating that the partial sequences of these HK genes originate from Cand. B. cysticola. The knowledge generated from the present study constitutes a major prerequisite for the future design of novel genotyping schemes for this bacterium.

Trichomoniasis, the most common non-viral sexually transmitted infection worldwide, is caused by the protozoon Trichomonas vaginalis. The 5- nitroimidazole drugs, of which metronidazole is the most prescribed, are the only effective drugs to treat trichomoniasis. Resistance against metronidazole is increasingly reported among T. vaginalis isolates. T. vaginalis can establish an endosymbiosis with two Mycoplasma species, Mycoplasma hominis and Candidatus Mycoplasma girerdii, whose presence has been demonstrated to influence several aspects of the protozoan pathobiology. The role of M. hominis in T. vaginalis resistance to metronidazole is controversial, while the influence of Ca. M. girerdii has never been investigated. In this work, we investigate the possible correlation between the presence of Ca. M. girerdii and/or M. hominis and the in vitro drug susceptibility in a large group of T. vaginalis isolated in Italy and in Vietnam. We also evaluated, via RNA-seq analysis, the expression of protozoan genes involved in metronidazole resistance in a set of syngenic T. vaginalis strains, differing only for the presence/absence of the two Mycoplasmas. Our results show that the presence of M. hominis significantly increases the sensitivity to metronidazole in T. vaginalis and affects gene expression. On the contrary, the symbiosis with Candidatus Mycoplasma girerdii seems to have no effect on metronidazole resistance in T. vaginalis.

Summary Huanglongbing (HLB) is currently considered the most destructive disease of citrus worldwide. In the major citrus‐growing areas in Asia and the US, the major causal agent of HLB is the bacterial pathogen Candidatus Liberibacter asiaticus (CLas). CLas is vectored by the Asian citrus psyllid, Diaphorina citri , in a persistent propagative manner. CLas cannot be cultured in vitro because of its unclear growth factors, leading to uncertainty in the infection mechanism of CLas at the cellular level in citrus and in D. citri . To characterize the detailed infection of CLas in the host and vector, the incidence of HLB was first investigated in citrus‐growing fields in Fujian Province, China. It was found that the positive association of the level of CLas infection in the leaves correlated with the symptoms. Then antibodies against peptides of the outer membrane protein (OMP) of CLas were prepared and tested. The antibodies OMP‐225, OMP‐333 and OMP724 showed specificity to citrus plants in western blot analyses, whereas the antibodies OMP‐47 and OMP‐225 displayed specificity to the D. citri vector. The application of OMP‐225 in the immunofluorescence assay indicated that CLas was located in and distributed throughout the phloem sieve cells of the leaf midribs and axile placenta of the fruit. CLas also infected the epithelial cells and visceral muscles of the alimentary canal of D. citri . The application of OMP‐333 in immunoelectron microscopy indicated the round or oval CLas in the sieve cells of leaf midribs and axile placenta of fruit as well as in the epithelial cells and reticular tissue of D. citri alimentary canal. These results provide a reliable means for HLB detection, and enlighten a strategy via neutralizing OMP to control HLB. These findings also provide insight for the further investigation on CLas infection and pathogenesis, as well as CLas–vector interaction.

Abstract“Candidatus Liberibacter asiaticus” (Ca. L. asiaticus) is the causal agent of Huanglongbing disease of citrus and current study focuses on the discovery of novel small‐molecule inhibitors against SecA protein of Ca. L. asiaticus. In this study, homologous modeling was used to construct the three‐dimensional structure of SecA. Then, molecular docking‐based virtual screening and two rounds of in vitro bacteriostatic experiments were utilized to identify novel small‐molecule inhibitors of SecA. Encouragingly, 93 compounds were obtained and two of them (P684‐2850, P684‐3808) showed strong antimicrobial activities against Liberibacter crescens BT‐1 in bacteriostatic experiments. Finally, molecular dynamics simulations were employed to explore the binding modes of the receptor–ligand complexes. Results in MD simulations showed that compound P684‐3808 was relatively stable during simulation, while compound P684‐2850 left the binding pocket. Compound P684‐3808 might be suitable as a lead compound for further development of antimicrobial compounds against SecA of Ca. L. asiaticus.