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ABSTRACTRickettsia rickettsii (R. rickettsii), the causative agent of Rocky Mountain spotted fever (RMSF), is the most pathogenic member among Rickettsia spp. Previous studies have shown that tripartite motif-containing 56 (TRIM56) E3 ligase-induced ubiquitination of STING is important for cytosolic DNA sensing and type I interferon production to induce anti-DNA viral immunity, but whether it affects intracellular replication of R. rickettsii remains uncharacterized. Here, we investigated the effect of TRIM56 on HeLa and THP-1 cells infected with R. rickettsii. We found that the expression of TRIM56 was upregulated in the R. rickettsii-infected cells, and the overexpression of TRIM56 inhibited the intracellular replication of R. rickettsii, while R. rickettsii replication was enhanced in the TRIM56-silenced host cells with the reduced phosphorylation of IRF3 and STING and the increased production of interferon-β. In addition, the mutation of the TRIM56 E3 ligase catalytic site impairs the inhibitory function against R. rickettsii in HeLa cells. Altogether, our study discovers that TRIM56 is a host restriction factor of R. rickettsii by regulating the cGAS-STING-mediated signaling pathway. This study gives new evidence for the role of TRIM56 in the innate immune response against intracellular bacterial infection and provides new therapeutic targets for RMSF.IMPORTANCEGiven that Rickettsia rickettsii (R. rickettsii) is the most pathogenic member within the Rickettsia genus and serves as the causative agent of Rocky Mountain spotted fever, there is a growing need to explore host targets. In this study, we examined the impact of host TRIM56 on R. rickettsii infection in HeLa and THP-1 cells. We observed a significant upregulation of TRIM56 expression in R. rickettsii-infected cells. Remarkably, the overexpression of TRIM56 inhibited the intracellular replication of R. rickettsii, while silencing TRIM56 enhanced bacterial replication accompanied by reduced phosphorylation of IRF3 and STING, along with increased interferon-β production. Notably, the mutation of the TRIM56’s E3 ligase catalytic site did not impede R. rickettsii replication in HeLa cells. Collectively, our findings provide novel insights into the role of TRIM56 as a host restriction factor against R. rickettsii through the modulation of the cGAS-STING signaling pathway.

Objectives: From March to June 2021, the reported number of clinically diagnosed endemic typhus in Anhui and Hubei provinces of China nearly increased four-fold compared with the monthly average numbers in last 5 years. An etiological and epidemiological investigation was initiated. Methods: The clinical specimens from the reported patients and the potential vector ticks were collected for molecular and serological detection, as well as cell culturing assay to identify the potential pathogen. Results: Polymerase chain reaction and sequence analysis of rrs and groEL showed that the pathogen from these patients was Ehrlichia sp., isolated from Haemaphysalis longicornis attached to these patients. The phylogenetic analysis based on 39 Ehrlichia genomes suggested that it should be taxonomically classified as a novel species, tentatively named “Candidatus Ehrlichia erythraense”. A total of 19 of 106 cases were confirmed as Candidatus Ehrlichia erythraense infections by polymerase chain reaction, sequencing, and/or serological tests. The most frequent symptoms were fever (100%), rashes (100%), asthenia (100%), anorexia (100%), and myalgia (79%). Conclusion: The occurrence of the disease presenting with fever and rashes in Anhui and Hubei provinces was caused by a novel species of the genus Ehrlichia; physicians need to be aware of this newly-discovered pathogen to ensure appropriate testing, treatment, and regional surveillance.

We report a case-series study of 5 patients with Japanese spotted fever from the Three Gorges Area in China, including 1 fatal case. Seroprevalence of Rickettsia japonica was ≈21% among the local population. Our report highlights the emerging potential threat to human health of Japanese spotted fever in the area.

Between November 2021 and January 2022, four patients of community-acquired pneumonia were admitted to the hospitals in Lishui city, Zhejiang province, China. Their main clinical manifestations were fever and dry cough as well as radiographic infiltrate, but the empiric antimicrobial therapy or traditional Chinese medicine was not effective for their illness. Clinical specimens from the patients as well as environmental and poultry specimens were collected for the determination of the causative pathogen. The ompA gene and seven housekeeping genes of Chlamydia psittaci were successfully amplified from all the patients, and the sequences of each gene were identical to one another, suggesting that they were infected by the same strain of C. psittaci. A novel strain of C. psittaci (LS strain) was isolated from the bronchoalveolar lavage fluid of patient 2 and its whole genome was obtained. Phylogenetic analyses based on the whole-genome sequences showed that the isolate is most closely related to the strain (WS/RT/E30) identified as genotype E/B. In addition, The ompA gene and four housekeeping genes of C. psittaci were also amplified from two of four faeces samples of geese at the home of patient 2, and the sequences from geese were 100% identical to those from the patients. Accordingly, these cases could be attributed to a circulating C. psittaci strain of genotype E/B in the local geese. Therefore, there is an urgent need to strengthen the regional surveillance on C. psittaci among poultry and humans for prevention and control of the outbreak of psittacosis in the city.

The order Rickettsiales in the class Alphaproteobacteria comprises vector-borne pathogens of both medical and veterinary importance. Ticks, as a group, are second only to mosquitoes as vectors of pathogens to humans, playing a critical role in the transmission of rickettsiosis. In the present study, 880 ticks collected from Jinzhai County, Lu'an City, Anhui Province, China in 2021–2022 were identified as belonging to five species from three genera. DNA extracted from individual ticks was examined using nested polymerase chain reaction targeting the 16S rRNA gene (rrs), and the gene fragments amplified were sequenced to detect and identify Rickettsiales bacteria in the ticks. For further identification, the rrs-positive tick samples were further amplified by PCR targeting the gltA and groEL gene and sequenced. As a result, 13 Rickettsiales species belonging to the genera Rickettsia, Anaplasma, and Ehrlichia were detected, including three tentative species of Ehrlichia. Our results reveal the extensive diversity of Rickettsiales bacteria in ticks from Jinzhai County, Anhui Province. There, emerging rickettsial species may be pathogenic and cause under-recognized diseases. Detection of several pathogens in ticks that are closely related to human diseases may indicate a potential risk of infection in humans. Therefore, additional studies to assess the potential public health risks of the Rickettsiales pathogens identified in the present study are warranted.

Abstract Background Coxiella burnetii (Cb) is the causative agent of the zoonotic disease Q fever which is distributed worldwide. Molecular typing of Cb strains is essential to find out the infectious source and prevent Q fever outbreaks, but there has been a lack of typing data for Cb strains in China. The aim of this study was to investigate the genotypes of Cb strains in wild rats in Yunnan Province, China. Results Eighty-six wild rats (Rattus flavipectus) were collected in Yunnan Province and 8 of the 86 liver samples from the wild rats were positive in Cb-specific quantitative PCR (qPCR). The Cb strains from the 8 rats were then typed into 3 genotypes using 10-spacer multispacer sequence typing (MST), and 2 of the 3 genotypes were recognized as novel ones. Moreover, the Cb strains in the wild rats were all identified as genotype 1 using 6-loci multilocus variable number of tandem repeat analysis (MLVA). Conclusions This is the first report of genotypic diversity of Cb strains from wild rats in China. Further studies are needed to explore the presence of more genotypes and to associate the genotypes circulating in the wildlife-livestock interaction with those causing human disease to further expand on the epidemiological aspects of the pathogen.

Coxiella burnetii is the etiological agent of the zoonotic disease Q fever, which is featured by its ability to replicate in acid vacuoles resembling the lysosomal network. One key virulence determinant of C. burnetii is the Dot/Icm system that transfers more than 150 effector proteins into host cells. These effectors function to construct the lysosome-like compartment permissive for bacterial replication, but the functions of most of these effectors remain elusive. In this study, we used an affinity tag purification mass spectrometry (AP-MS) approach to generate a C. burnetii-human protein-protein interaction (PPI) map involving 53 C. burnetii effectors and 3480 host proteins. This PPI map revealed that the C. burnetii effector CBU0425 (designated CirB) interacts with most subunits of the 20S core proteasome. We found that ectopically expressed CirB inhibits hydrolytic activity of the proteasome. In addition, overexpression of CirB in C. burnetii caused dramatic inhibition of proteasome activity in host cells, while knocking down CirB expression alleviated such inhibitory effects. Moreover, we showed that a region of CirB that spans residues 91-120 binds to the proteasome subunit PSMB5 (beta 5). Finally, PSMB5 knockdown promotes C. burnetii virulence, highlighting the importance of proteasome activity modulation during the course of C. burnetii infection.

Chlamydia psittaci is the causative agent of psittacosis, a worldwide zoonotic disease. A rapid, specific, and sensitive diagnostic assay would be benefit for C. psittaci infection control. In this study, an assay combining recombinase-aided amplification and a lateral flow strip (RAA-LF) for the detection of active C. psittaci infection was developed. The RAA-LF assay targeted the CPSIT_RS02830 gene of C. psittaci and could be accomplished in 15 min at a single temperature (39°C). The analytical sensitivity of the assay was as low as 1 × 100 copies/μl and no cross-reaction with some other intracellular pathogens was observed. Moreover, all feces samples from mice infected with C. psittaci at day-1 post-infection were positive in the RAA-LF assay. In conclusion, the RAA-LF assay provides a convenient, rapid, specific and sensitive method for detection of active C. psittaci infection and it is also suitable for C. psittaci detection in field.

Rhipicephalus microplus, a vector that can transmit many pathogens to humans and domestic animals, is widely distributed in Yunnan province, China. However, few reports on the prevalence of tick-borne pathogens (TBPs) in Rh. microplus in Yunnan are available. The aim of this study was to detect TBPs in Rh. microplus in Yunnan and to analyze the phylogenetic characterization of TBPs detected in these ticks. The adult Rh. microplus (n = 516) feeding on cattle were collected. The pooled DNA samples of these ticks were evaluated using metagenomic next-generation sequencing (mNGS) and then TBPs in individual ticks were identified using genus- or group-specific nested polymerase chain reaction (PCR) combined with DNA sequencing assay. As a result, Candidatus Rickettsia jingxinensis (24.61%, 127/516), Anaplasma marginale (13.18%, 68/516), Coxiella burnetii (3.10%, 16/516), and Coxiella-like endosymbiont (CLE) (8.33%, 43/516) were detected. The dual coinfection with Ca. R. jingxinensis and A. marginale and the triple coinfection with Ca. R. jingxinensis, A. marginale, and CLE were most frequent and detected in 3.68% (19/516) and 3.10% (16/516) of these ticks, respectively. The results provide insight into the diversity of TBPs and their coinfections in Rh. microplus in Yunnan province of China, reporting for the first time that C. burnetii had been found in Rh. microplus in China. Multilocus variable number tandem repeat analysis with 6 loci (MLVA-6) discriminated the C. burnetii detected in Rh. microplus in Yunnan into MLVA genotype 1, which is closely related to previously described genotypes found primarily in tick and human samples from different regions of the globe, indicating a potential public health threat posed by C. burnetii in Rh. microplus in Yunnan.

Hyalomma marginatum is an important arthropod vector in the transmission of various zoonoses. The aim of this study was to identify the tick-borne pathogens (TBPs) maintained in Hy. marginatum in Tibet and to estimate the risk of human tick-borne diseases. Adult Hy. marginatum ticks (n = 14) feeding on yaks were collected. The individual DNA samples of these ticks were sequenced with metagenomic next-generation sequencing to survey the presence of TBPs. TBPs in individual ticks were identified with nested polymerase chain reaction (PCR) combined with DNA sequencing. The presence of Rickettsia , Anaplasma , and Ehrlichia in individual ticks was indicated by the taxonomic profiles at the genus level, but only Rickettsia aeschlimannii (100%, 13/13) was further detected in the ticks by nested PCR. This study provides information on the microbial communities of Hy. marginatum in Tibet, China, and provides the first report of R. aeschlimannii found in Hy. marginatum in Tibet. The results of this study indicated that yaks in Tibet are exposed to R. aeschlimannii .