Obligate Intracellular Bacterium Research Articles

Benidipine impairs innate immunity converting sublethal to lethal infections in a murine model of spotted fever rickettsiosis.

Spotted fever group rickettsiae are tick-borne obligate intracellular bacteria that infect microvascular endothelial cells. Humans and mammalian infection results in endothelial cell barrier dysfunction and increased vascular permeability. We previously demonstrated that treatment of Rickettsia parkeri-infected cells with the calcium channel blocker benidipine significantly delayed vascular barrier permeability. Thus, we hypothesized that benidipine, known to be safe and effective for other clinical processes, could reduce rickettsia-induced vascular permeability in vivo in an animal model of spotted fever rickettsiosis. Based on liver, lung and brain vascular FITC-dextran extravasation studies, benidipine did not reliably impact vascular permeability. However, it precipitated a deleterious effect on responses to control sublethal R. parkeri infection. Animals treated with benidipine alone had no clinical signs or changes in histopathology and splenic immune cell distributions. Benidipine-treated infected animals had marked increases in tissue and blood bacterial loads, more extensive inflammatory histopathologic injury, and changes in splenic architecture and immune cell distributions potentially reflecting diminished Ca2+ signaling, reduced innate immune cell activation, and loss of rickettsial propagation control. Impaired T cell activation by R. parkeri antigen in the presence of benidipine was confirmed in vitro with the use of NKT cell hybridomas. The unexpected findings stand in stark contrast to recent discussions of the benefits of calcium channel blockers for viral infections and chronic infectious or inflammatory diseases. A role for calcium channel blockers in exacerbation of human rickettsiosis and acute inflammatory infections should be evaluated by a retrospective review of patient's outcomes and medications.

Improved metagenome assemblies through selective enrichment of bacterial genomic DNA from eukaryotic host genomic DNA using ATAC-seq.

Genomics can be used to study the complex relationships between hosts and their microbiota. Many bacteria cannot be cultured in the laboratory, making it difficult to obtain adequate amounts of bacterial DNA and to limit host DNA contamination for the construction of metagenome-assembled genomes (MAGs). For example, Wolbachia is a genus of exclusively obligate intracellular bacteria that live in a wide range of arthropods and some nematodes. While Wolbachia endosymbionts are frequently described as facultative reproductive parasites in arthropods, the bacteria are obligate mutualistic endosymbionts of filarial worms. Here, we achieve 50-fold enrichment of bacterial sequences using ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) with Brugia malayi nematodes, containing Wolbachia (wBm). ATAC-seq uses the Tn5 transposase to cut and attach Illumina sequencing adapters to accessible DNA lacking histones, typically thought to be open chromatin. Bacterial and mitochondrial DNA in the lysates are also cut preferentially since they lack histones, leading to the enrichment of these sequences. The benefits of this include minimal tissue input (<1 mg of tissue), a quick protocol (<4 h), low sequencing costs, less bias, correct assembly of lateral gene transfers and no prior sequence knowledge required. We assembled the wBm genome with as few as 1 million Illumina short paired-end reads with >97% coverage of the published genome, compared to only 12% coverage with the standard gDNA libraries. We found significant bacterial sequence enrichment that facilitated genome assembly in previously published ATAC-seq data sets from human cells infected with Mycobacterium tuberculosis and C. elegans contaminated with their food source, the OP50 strain of E. coli. These results demonstrate the feasibility and benefits of using ATAC-seq to easily obtain bacterial genomes to aid in symbiosis, infectious disease, and microbiome research.

Pathogenic Rickettsia spp. as emerging models for bacterial biology.

Our understanding of free-living bacterial models like Escherichia coli far outpaces that of obligate intracellular bacteria, which cannot be cultured axenically. All obligate intracellular bacteria are host-associated, and many cause serious human diseases. Their constant exposure to the distinct biochemical niche of the host has driven the evolution of numerous specialized bacteriological and genetic adaptations, as well as innovative molecular mechanisms of infection. Here, we review the history and use of pathogenic Rickettsia species, which cause an array of vector-borne vascular illnesses, as model systems to probe microbial biology. Although many challenges remain in our studies of these organisms, the rich pathogenic and biological diversity of Rickettsia spp. constitutes a unique backdrop to investigate how microbes survive and thrive in host and vector cells. We take a bacterial-focused perspective and highlight emerging insights that relate to new host-pathogen interactions, bacterial physiology, and evolution. The transformation of Rickettsia spp. from pathogens to models demonstrates how recalcitrant microbes may be leveraged in the lab to tap unmined bacterial diversity for new discoveries. Rickettsia spp. hold great promise as model systems not only to understand other obligate intracellular pathogens but also to discover new biology across and beyond bacteria.

Molecular epidemiology of Coxiella burnetii infection in sheep and goats in Henan province, China

Q fever is a significant zoonotic disease caused by Coxiella burnetii, an obligate intracellular gram-negative bacterium. Although C. burnetii infection has been identified in various animal species, domestic ruminants serve as the primary reservoirs and main sources of human infection. Understanding of the epidemiology of C. burnetii in domestic ruminants is crucial for preventing and controlling of C. burnetii infection in humans. In this study, spleen tissues from sheep and goats were collected in Hennan province, China. Through PCR screening, C. burnetii was detected in sheep and goats in Henan province with an overall infection rate of 6.8%. Sequence comparison and phylogenetic analysis revealed that all newly identified C. burnetii strains shared a close genetic relationship with those found in humans worldwide. These findings highlight the high risk of C. burnetii infection among slaughterhouse workers and emphasize the importance of epidemiological studies that investigate samples from both humans and animals within the "One Health" framework. Such surveillance will contribute to a better understanding of the epidemic situation and aid in the development of effective prevention and control strategies for C. burnetii infections in humans.

Expression activation of over 70% of Chlamydia trachomatis genes during the first hour of infection.

The obligate intracellular bacterium Chlamydia has a unique developmental cycle that alternates between two contrasting cell types. With a hardy envelope and highly condensed genome, the small elementary body (EB) maintains limited metabolic activities yet survives in extracellular environments and is infectious. After entering host cells, EBs differentiate into larger and proliferating reticulate bodies (RBs). Progeny EBs are derived from RBs in late developmental stages and eventually exit host cells. How expression of the chlamydial genome consisting of nearly 1,000 genes governs the chlamydial developmental cycle is unclear. A previous microarray study identified only 29 Chlamydia trachomatis immediate early genes, defined as genes with increased expression during the first hour postinoculation in cultured cells. In this study, we performed more sensitive RNA sequencing (RNA-Seq) analysis for C. trachomatis cultures with high multiplicities of infection. Remarkably, we observed well over 700 C. trachomatis genes that underwent 2- to 900-fold activation within 1 hour postinoculation. Quantitative reverse transcription real-time PCR analysis was further used to validate the activated expression of a large subset of the genes identified by RNA-Seq. Importantly, our results demonstrate that the immediate early transcriptome is over 20 times more extensive than previously realized. Gene ontology analysis indicates that the activated expression spans all functional categories. We conclude that over 70% of C. trachomatis genes are activated in EBs almost immediately upon entry into host cells, thus implicating their importance in initiating rapid differentiation into RBs and establishing an intracellular niche conducive with chlamydial development and growth.

Aptamer selection against cell extracts containing the zoonotic obligate intracellular bacterium, Anaplasma phagocytophilum

A. phagocytophilum is a zoonotic and tick-borne bacterium, threatening human and animal health. Many questions persist concerning the variability of strains and the mechanisms governing the interactions with its different hosts. These gaps can be explained by the difficulty to cultivate and study A. phagocytophilum because of its strict intracellular location and the lack of specific tools, in particular monoclonal antibodies, currently unavailable. The objective of our study was to develop DNA aptamers against A. phagocytophilum, or molecules expressed during the infection, as new study and/or capture tools. Selecting aptamers was a major challenge due to the strict intracellular location of the bacterium. To meet this challenge, we set up a customized selection protocol against an enriched suspension of A. phagocytophilum NY18 strain, cultivated in HL-60 cells. The implementation of SELEX allowed the selection of three aptamers, characterized by a high affinity for HL-60 cells infected with A. phagocytophilum NY18 strain. Interestingly, the targets of these three aptamers are most likely proteins expressed at different times of infection. The selected aptamers could contribute to increase our understanding of the interactions between A. phagocytophilum and its hosts, as well as permit the development of new diagnostic, therapeutic or drug delivery appliances.

TREATMENT OF INFECTION OF EHRLICHIA SP. IN A MIX BREED DOG

Ehrlichiosis is a disease in dogs caused by the bacterium Ehrlichia sp. and affects all ages and breeds. Ehrlichia sp. is an obligate intracellular bacterium, which means that this bacterium is able to enter into its host cells and its life depends on its host. This article was created to provide information regarding the management of Ehrlichia infection in dogs. The case animal is a mixed breed dog named Cio, 2 years old, female, weighing 11 kg. The dog come with complaints of itching and hair loss. The dog has a history of blood parasites (Ehrlichiosis) a year ago and has already been to the clinic. The blood test results found Ehrlichia sp and the test kit was positive. Based on the anamnesis, and the results of clinical and supporting examinations, the dog was diagnosed with ehrlichiosis with Rhipicephalus sanguineus tick infestation. The therapy provided includes symptomatic therapy, causative therapy, and supportive therapy. Diphenhydramine HCl injection 1.1 mL, intramuscularly. The antibiotic doxycycline is given at a dose of 10 mg/kg, once a day for 28 days. Sangobion was given to reduce clinical symptoms, which is anemia and thrombocytopenia. Dog was bathed with a Tick and Flea shampoo which contains insecticides (Pyrethrin and Piperonyl). The evaluation was carried out after the antibiotics were finished, based on the owner's statement, the condition of the case dog was healthy, no longer scratching and falling hair, and the tick infestation had disappeared. It is recommended to eradicate the vector (Rhipicephalus sanguineus) in the environment where animals live. This is done to prevent recurrent infections from Ehrlichia.

MICROBIAL DRIVERS OF REGION-SPECIFIC SUSCEPTIBILITY TO CHRONIC, TNF-INDUCED INTESTINAL INFLAMMATION

Abstract BACKGROUND The majority of Crohn’s disease patients present with ileitis or ileocolitis, however, the factors that drive the development of inflammation in these different sites remain elusive. The TnfΔARE mouse model recapitulates many features of Crohn’s disease and is driven by systemic overexpression of TNF. AIMS To-date, the TnfΔARE model is published as a spontaneous intestinal inflammation model that is specific to the ileum, however, we observe severe colonic inflammation that is dependent on the murine housing facility. We aimed to use experimental approaches to investigate the epithelial and microbial factors that drive ileal and colonic inflammation in the TnfΔARE model of Crohn’s disease. METHODS Tnf ΔARE mice were housed in two murine housing facilities, termed “restrictive” and “permissive”. Pathological scoring was used to assess intestinal inflammation in TnfΔARE mice. Murine co-housing methods were used to expose non-colitic mice to those with colitis. Shotgun metagenomics was performed to determine differences in the intestinal microbiota between the two murine housing facilities. Doxycycline treatment was used to treat Chlamydia muridarum (CM) infection in mice. Various in vivo and in vitro perturbations were performed to test the role of intestinal epithelial cell expression of indoleamine 2,3-dioxygenase (IDO1) in ileal and colonic inflammation. Human single-cell RNA-sequencing (scRNA-seq) data, along with tissue samples, were analyzed to determine whether epithelial cell signaling pathways and microbiota were conserved in Crohn’s disease patients. RESULTS Tnf ΔARE mice from the restrictive murine housing facility developed the expected phenotype of ileitis, while TnfΔARE mice from the permissive murine housing facility developed ileocolitis. Upon co-housing, restrictive facility TnfΔARE mice develop ileocolitis. Permissive facility mice, but not restrictive facility mice, harbored the obligate intracellular bacterium CM in proximal colonic epithelia. Doxycycline treatment effectively cleared CM colonization and was associated with a reduction in colonic inflammation. IDO1 expression was upregulated in intestinal epithelial secretory cells in CM-infected mice and perturbation of this pathway resulted in a reduction of intestinal inflammation. Analysis of patient scRNA-seq and tissue samples revealed IDO1-expressing epithelial cells in the ascending colon and the presence of epithelia-associated microbiota. CONCLUSION Here, we associate CM infection with the development of ileocolitis the TnfΔARE mouse model of Crohn’s disease. We reveal the role of IDO1-expressing intestinal secretory cells in TnfΔARE mice with and without colonic involvement. Finally, we demonstrate conservation of these pathways and microbiota in human Crohn’s disease samples.

Targeted repression of topA by CRISPRi reveals a critical function for balanced DNA topoisomerase I activity in the Chlamydia trachomatis developmental cycle.

Chlamydia trachomatis is an obligate intracellular bacterium that is responsible for the most prevalent bacterial sexually transmitted infection. Changes in DNA topology in this pathogen have been linked to its pathogenicity-associated developmental cycle. Here, evidence is provided that the balanced activity of DNA topoisomerases contributes to controlling Chlamydia developmental processes. Utilizing catalytically inactivated Cas12 (dCas12)-based clustered regularly interspaced short palindromic repeats interference (CRISPRi) technology, we demonstrate targeted knockdown of chromosomal topA transcription in C. trachomatis without detected toxicity of dCas12. Repression of topA impaired the developmental cycle of C. trachomatis mostly through disruption of its differentiation from a replicative form to an infectious form. Consistent with this, expression of late developmental genes of C. trachomatis was downregulated, while early genes maintained their expression. Importantly, the developmental defect associated with topA knockdown was rescued by overexpressing topA at an appropriate degree and time, directly linking the growth patterns to the levels of topA expression. Interestingly, topA knockdown had effects on DNA gyrase expression, indicating a potential compensatory mechanism for survival to offset TopA deficiency. C. trachomatis with topA knocked down displayed hypersensitivity to moxifloxacin that targets DNA gyrase in comparison with the wild type. These data underscore the requirement of integrated topoisomerase actions to support the essential developmental and transcriptional processes of C. trachomatis.IMPORTANCEWe used genetic and chemical tools to demonstrate the relationship of topoisomerase activities and their obligatory role for the chlamydial developmental cycle. Successfully targeting the essential gene topA with a CRISPRi approach, using dCas12, in C. trachomatis indicates that this method will facilitate the characterization of the essential genome. These findings have an important impact on our understanding of the mechanisms by which well-balanced topoisomerase functions in adaptation of C. trachomatis to unfavorable growth conditions imposed by antibiotics.

Opening the Black Box of Host Range, Vectorial Diversity, and Genetic Variants of Genus Anaplasma: The Contributing Factors Toward Its Zoonosis.

Background: Genus Anaplasma of the family Anaplasmataceae possesses bacteria of hematopoietic origin, which are obligate intracellular Gram-negative bacteria transmitted mainly by tick vectors. The members of this group of infectious agents are not new as etiological agents of animal diseases worldwide. However, now, reports of their zoonotic potential have gained currency to study these pathogens. The emergence of new species of Anaplasma and the spread of existing species to new areas and hosts highlight the importance of monitoring and improving diagnostic and treatment options for zoonotic diseases caused by Anaplasma. Conclusion: This review focuses on the general and distinctive characteristics of Anaplasma spp., with particular emphasis on the novel species and their diverse spectrum of hosts as potential risk factors impacting its emerging zoonosis.

The Chlamydia-related Waddlia chondrophila encodes functional type II toxin-antitoxin systems.

Bacterial toxin-antitoxin (TA) systems are widespread in chromosomes and plasmids of free-living microorganisms, but only a few have been identified in obligate intracellular species. We found seven putative type II TA modules in Waddlia chondrophila, a Chlamydia-related species that is able to infect a very broad series of eukaryotic hosts, ranging from protists to mammalian cells. The RNA levels of Waddlia TA systems are significantly upregulated by iron starvation and novobiocin, but they are not affected by antibiotics such as β-lactams and glycopeptides, which suggests different mechanisms underlying stress responses. Five of the identified TA modules, including HigBA1 and MazEF1, encoded on the Waddlia cryptic plasmid, proved to be functional when expressed in a heterologous host. TA systems have been associated with the maintenance of mobile genetic elements, bacterial defense against bacteriophages, and persistence upon exposure to adverse conditions. As their RNA levels are upregulated upon exposure to adverse conditions, Waddlia TA modules may be involved in survival to stress. Moreover, as Waddlia can infect a wide range of hosts including free-living amoebae, TA modules could also represent an innate immunity system to fight against bacteriophages and other microorganisms with which Waddlia has to share its replicative niche.IMPORTANCEThe response to adverse conditions, such as exposure to antibiotics, nutrient starvation and competition with other microorganisms, is essential for the survival of a bacterial population. TA systems are modules composed of two elements, a toxic protein and an antitoxin (protein or RNA) that counteracts the toxin. Although many aspects of TA biological functions still await to be elucidated, TAs have often been implicated in bacterial response to stress, including the response to nutrient starvation, antibiotic treatment and bacteriophage infection. TAs are ubiquitous in free-living bacteria but rare in obligate intracellular species such as chlamydiae. We identified functional TA systems in Waddlia chondrophila, a chlamydial species with a strikingly broad host range compared to other chlamydiae. Our work contributes to understand how obligate intracellular bacteria react to adverse conditions that might arise from competition with other viruses/bacteria for the same replicative niche and would threaten their ability to replicate.

Lysosomal trafficking regulator restricts intracellular growth of Coxiella burnetii by inhibiting the expansion of Coxiella-containing vacuole and upregulating nos2 expression.

Coxiella burnetii is an obligate intracellular bacterium that causes Q fever, a zoonotic disease typically manifests as a severe flu-illness. After invading into the host cells, C. burnetii delivers effectors to regulate the vesicle trafficking and fusion events to form a large and mature Coxiella-containing vacuole (CCV), providing sufficient space and nutrition for its intracellular growth and proliferation. Lysosomal trafficking regulator (LYST) is a member of the Beige and Chediak-Higashi syndrome (BEACH) family, which regulates the transport of vesicles to lysosomes and regulates TLR signaling pathway, but the effect of LYST on C. burnetii infection is unclear. In this study, a series of experiments has been conducted to investigate the influence of LYST on intracellular growth of C. burnetii. Our results showed that lyst transcription was up-regulated in the host cells after C. burnetii infection, but there is no significant change in lyst expression level after infection with the Dot/Icm type IV secretion system (T4SS) mutant strain, while CCVs expansion and significantly increasing load of C. burnetii appeared in the host cells with a silenced lyst gene, suggesting LYST inhibits the intracellular proliferation of C. burnetii by reducing CCVs size. Then, the size of CCVs and the load of C. burnetii in the HeLa cells pretreated with E-64d were significantly decreased. In addition, the level of iNOS was decreased significantly in LYST knockout THP-1 cells, which was conducive to the intracellular replication of C. burnetii. This data is consistent with the phenotype of L-NMMA-treated THP-1 cells infected with C. burnetii. Our results revealed that the upregulation of lyst transcription after infection is due to effector secretion of C. burnetii and LYST inhibit the intracellular replication of C. burnetii by reducing the size of CCVs and inducing nos2 expression.

Critical roles of Rickettsia parkeri outer membrane protein B (OmpB) in the tick host.

Rickettsia parkeri is a pathogen of public health concern and transmitted by the Gulf Coast tick, Amblyomma maculatum. Rickettsiae are obligate intracellular bacteria that enter and replicate in diverse host cells. Rickettsial outer membrane protein B (OmpB) functions in bacterial adhesion, invasion, and avoidance of cell-autonomous immunity in mammalian cell infection, but the function of OmpB in arthropod infection is unknown. In this study, the function of R. parkeri OmpB was evaluated in the tick host. R. parkeri wild-type and R. parkeri ompBSTOP::tn (non-functional OmpB) were capillary fed to naïve A. maculatum ticks to investigate dissemination in the tick and transmission to vertebrates. Ticks exposed to R. parkeri wild-type had greater rickettsial loads in all organs than ticks exposed to R. parkeri ompBSTOP::tn at 12 h post-capillary feeding and after 1 day of feeding on host. In rats that were exposed to R. parkeri ompBSTOP::tn-infected ticks, dermal inflammation at the bite site was less compared to R. parkeri wild-type-infected ticks. In vitro, R. parkeri ompBSTOP::tn cell attachment to tick cells was reduced, and host cell invasion of the mutant was initially reduced but eventually returned to the level of R. parkeri wild-type by 90 min post-infection. R. parkeri ompBSTOP::tn and R. parkeri wild-type had similar growth kinetics in the tick cells, suggesting that OmpB is not essential for R. parkeri replication in tick cells. These results indicate that R. parkeri OmpB functions in rickettsial attachment and internalization to tick cells and pathogenicity during tick infection.

Enhancement of Macrophage Immunity against Chlamydial Infection by Natural Killer T Cells.

Lung macrophage (LM) is vital in host defence against bacterial infections. However, the influence of other innate immune cells on its function, including the polarisation of different subpopulations, remains poorly understood. This study examined the polarisation of LM subpopulations (monocytes/undifferentiated macrophages (Mo/Mφ), interstitial macrophages (IM), and alveolar macrophages (AM)). We further assessed the effect of invariant natural killer T cells (iNKT) on LM polarisation in a protective function against Chlamydia muridarum, an obligate intracellular bacterium, and respiratory tract infection. We found a preferentially increased local Mo/Mφ and IMs with a significant shift to a type-1 macrophage (M1) phenotype and higher expression of iNOS and TNF-α. Interestingly, during the same infection, the alteration of macrophage subpopulations and the shift towards M1 was much less in iNKT KO mice. More importantly, functional testing by adoptively transferring LMs isolated from iNKT KO mice (iNKT KO-Mφ) conferred less protection than those isolated from wild-type mice (WT-Mφ). Further analyses showed significantly reduced gene expression of the JAK/STAT signalling pathway molecules in iNKT KO-Mφ. The data show an important role of iNKT in promoting LM polarisation to the M1 direction, which is functionally relevant to host defence against a human intracellular bacterial infection. The alteration of JAK/STAT signalling molecule gene expression in iNKT KO-Mφ suggests the modulating effect of iNKT is likely through the JAK/STAT pathway.

Chlamydiae in corals: shared functional potential despite broad taxonomic diversity.

Cnidarians, such as corals and sea anemones, associate with a wide range of bacteria that have essential functions, including nutrient cycling and the production of antimicrobial compounds. Within cnidarians, bacteria can colonize all microhabitats including the tissues. Among them are obligate intracellular bacteria of the phylum Chlamydiota (chlamydiae) whose impact on cnidarian hosts and holobionts, especially corals, remain unknown. Here, we conducted a meta-analysis of previously published 16S rRNA gene metabarcoding data from cnidarians (e.g. coral, jellyfish, and anemones), eight metagenome-assembled genomes (MAGs) of coral-associated chlamydiae, and one MAG of jellyfish-associated chlamydiae to decipher their diversity and functional potential. While the metabarcoding dataset showed an enormous diversity of cnidarian-associated chlamydiae, six out of nine MAGs were affiliated with the Simkaniaceae family. The other three MAGs were assigned to the Parasimkaniaceae, Rhabdochlamydiaceae, and Anoxychlamydiaceae, respectively. All MAGs lacked the genes necessary for an independent existence, lacking any nucleotide or vitamin and most amino acid biosynthesis pathways. Hallmark chlamydial genes, such as a type III secretion system, nucleotide transporters, and genes for host interaction, were encoded in all MAGs. Together these observations suggest an obligate intracellular lifestyle of coral-associated chlamydiae. No unique genes were found in coral-associated chlamydiae, suggesting a lack of host specificity. Additional studies are needed to understand how chlamydiae interact with their coral host, and other microbes in coral holobionts. This first study of the diversity and functional potential of coral-associated chlamydiae improves our understanding of both the coral microbiome and the chlamydial lifestyle and host range.

Vitamin D effects on Chlamydia trachomatis infection: a case-control and experimental study.

Vitamin D deficiency is the most common nutritional deficiency worldwide. Chronic vitamin D deficiency causes immune system dysfunction, which increases susceptibility to pathogens such as bacteria, especially intracellular parasites, and viruses. Chlamydia trachomatis (C. t) is an obligate intracellular parasitic bacterium that causes a variety of sequelae. We speculated that vitamin D might be associated with C. t infection. This study aimed to address this gap in knowledge by investigating the relationship between vitamin D and C. t infection using both in vitro and in vivo models. The addition of calcitriol to McCoy cell culture in vitro delayed and reduced the quantity and volume of inclusions compared to the control group. Macrophages of peritoneally lavaged mice co-cultured with McCoy decreased the infection rate and delayed the appearance of inclusions. In mice models of vitamin D deficiency, mice in the VD-group exhibited more severe genital tract inflammation and a longer duration of infection after inoculation with C. t in the genital tract. Supplementing these mice with vitamin D3 during treatment enhanced the therapeutic effect of antibiotics. We also conducted a case-control study involving 174C. t-positive patients (95 males and 79 females) and 380 healthy volunteers (211 males and 169 females) aged 20-49 from January 2016 to March 15, 2017. Serum 25-(OH)D concentration was measured by assessing morning fasting blood samples of healthy volunteers and C. t-positive patients 1 day before antibiotic treatment and the next day after one course of treatment. The patients were followed up for 1 month and evaluated for recovery. The results showed that vitamin D deficiency was a risk factor for C. t infection and treatment failure. In summary, findings from experimental and clinical studies indicate a close association between vitamin D levels and C. t infection and treatment outcomes. Given the affordability and safety of vitamin D, both healthy individuals and patients should focus on vitamin D intake. Vitamin D supplementation could enhance treatment success and should be used as an adjunctive therapy alongside antibiotic therapy for C. t infections, pending confirmation in larger, prospective, randomized controlled trials.

Diagnosis and treatment of Anaplasma centrale in a domestic cat at Animal Hospital of Hasanuddin University Makassar

Anaplasmosis is a disease caused by obligate gram-negative intracellular bacteria. Anaplasma in cats is generally infected by Anaplasma phagocytophilum, however there are very limited reports of Anaplasma bovis such as Anaplasma marginale and Anaplasma centrale being reported to infect cats. This is the first report of anaplasmosis caused by Anaplasma centrale in cats. The owner brought a one-year-old female domestic cat to the Animal Hospital of Hasanuddin University in Makassar with the condition of not wanting to eat, weak, lethargic, dehydrated, and not defaecating. Diagnosis is carried out through anamnesis, physical examination, clinical examination, and laboratory examination, in the form of haematology and blood smear tests. The results of the examination revealed flea infestation, a blood test identified Anaplasma centrale with characteristics such as the presence of morulae in the middle part of the erythrocytes, and routine blood work showed anaemia which was seen in the MCV parameters increasing and the MCHC values decreased. Treatment consists of administration of antibiotics, anti-inflammatory drugs, and supportive therapy. The cat showed good results, with symptoms starting to disappear and the animal appeared active.

Lawsonia intracellularis flagellin protein LfliC stimulates NF-κB and MAPK signaling pathways independently of TLR5 interaction

Lawsonia intracellularis, a Gram-negative obligate intracellular bacterium and etiologic agent of porcine proliferative enteropathy, was observed to have a long, single, and unipolar flagellum. Bacterial flagellar filament comprises thousands of copies of the protein flagellin (FliC), and has been reported to be recognized by Toll-like receptor (TLR5) to activate the NF-κB and MAPK signaling pathways, thereby inducing the expression of proinflammatory genes. Recently, two L. intracellularis flagellin proteins, LfliC and LFliC, were reported to be involved in bacterial-host interaction and immune response. Here, to further explore the role of LfliC in proinflammatory response, we purified LfliC, and found that its exposure could activate NF-κB signaling pathway in both HEK293T and IPI-FX cells, as well as activate MAPK p38 and ERK1/2 in HEK293T cells but not in IPI-FX cells. However, our yeast two-hybrid and co-immunoprecipitation assay results revealed that LfliC has no interaction with the porcine TLR5 ECD domain though it harbors the conserved D1-like motif required for the interaction. Moreover, LfliC was identified as a substrate of the virulence-associated type III secretion system (T3SS) by using the heterologous Y. enterocolitica system. Transient expression of LfliC also activated the NF-κB and MAPK signaling pathway in HEK293T cells. Collectively, our results suggest that both the exposure and expression of L. intracellularis LfliC can induce the NF-κB and MAPK signaling pathway in mammalian cells. Our findings may provide important implications and resources for the development of diagnostic tools or vaccines and dissection of the pathogenesis of L. intracellularis.

Requirement of GrgA for Chlamydia infectious progeny production, optimal growth, and efficient plasmid maintenance.

Hallmarks of the developmental cycle of the obligate intracellular pathogenic bacterium Chlamydia are the primary differentiation of the infectious elementary body (EB) into the proliferative reticulate body (RB) and the secondary differentiation of RBs back into EBs. The mechanisms regulating these transitions remain unclear. In this report, we developed an effective novel strategy termed dependence on plasmid-mediated expression (DOPE) that allows for the knockdown of essential genes in Chlamydia. We demonstrate that GrgA, a Chlamydia-specific transcription factor, is essential for the secondary differentiation and optimal growth of RBs. We also show that GrgA, a chromosome-encoded regulatory protein, controls the maintenance of the chlamydial virulence plasmid. Transcriptomic analysis further indicates that GrgA functions as a critical regulator of all three sigma factors that recognize different promoter sets at developmental stages. The DOPE strategy outlined here should provide a valuable tool for future studies examining chlamydial growth, development, and pathogenicity.

Xác định thành phần loài Rickettsia trên chuột và một số ngoại ký sinh trùng ở khu vực đóng quân một số đơn vị quân đội miền Bắc

Rickettsia, a gram-negative bacterium, and an obligate intracellular bacterium, can potentially cause fatal acute fever if not promptly detected and treated. It's often transmitted to humans by arthropods like ticks, fleas, and lice. Rodents and small mammals can serve as key hosts for various Rickettsia species. Our study aimed to identify Rickettsia species using nested PCR and gene sequencing of ompA, ompB, 17-kDa, and gltA genes based on 29 DNA samples, which tested positive for Rickettsia spp collected from mice, ticks, fleas, and lice at military units in Lang Son, Nghe An, Hai Duong, and Bac Giang, The phylogenetic analysis showed that 9/29 (31%) of Rickettsia samples belonged to Rickettsia felis species, 1/29 (3.5%) of Rickettsia samples exhibited a 99.5% ompB gene sequence similarity to Rickettsia heilongjiangensis species .