Obligate Intracellular Bacterium Research Articles

Rab27a via Its effector JFC1 Localizes to Anaplasma Inclusions and Promotes Anaplasma Proliferation in Leukocytes

Anaplasma phagocytophilum is an obligatory intracellular bacterium that causes tick-borne zoonosis called human granulocytic anaplasmosis. Mechanisms by which Anaplasma replicates inside of the membrane-bound compartment called “inclusion” in neutrophils is incompletely understood. A small GTPase Rab27a is found in the secretory granules and multivesicular endosomes. In this study we found Rab27a-containing granules were localized to Anaplasma inclusions in guanine nucleotide-dependent manner, and constitutively active Rab27a enhanced Anaplasma infection and dominant-negative Rab27a inhibited Anaplasma infection. Rab27a effector, JFC1 is known to mediate docking/fusion of Rab27a-bearing granules for exocytosis in leukocytes. shRNA stable knockdown of Rab27a or JFC1 inhibited Anaplasma infection in HL-60 cells. Similar to Rab27a, JFC1 were localized to Anaplasma inclusions by immunostaining, and fluorescent probe transfection in live cells. The JFC1 C2A domain that binds 3'-phosphoinositides, was sufficient and required for JFC1 and Rab27a localization to Anaplasma inclusions which were enriched with phosphatidylinositol 3-phosphate. Nexinhib20, the small molecule inhibitor specific to Rab27a and JFC1 binding, inhibited Anaplasma infection. Taken together, these results imply elevated phosphatidylinositol 3-phosphate in the inclusion membrane recruits JFC1 to mediate Rab27a-bearing granules/vesicles to dock/fuse with Anaplasma inclusions, the lumen of which is topologically equivalent to the exterior of the cell to benefit Anaplasma proliferation.

Draft genomes of novel avian Chlamydia abortus strains from Australian Torresian crows (Corvus orru) shed light on possible reservoir hosts and evolutionary pathways.

Chlamydia abortus, an obligate intracellular bacterium, is a major causative agent of reproductive loss in ruminants, with zoonotic potential. Though this pathogen is primarily known to infect livestock, recent studies have detected and isolated genetically distinct avian strains of C. abortus from wild birds globally. Before this study, only five avian C. abortus genomes were publicly available. Therefore, we performed culture-independent probe-based whole-genome sequencing on clinical swabs positive for avian C. abortus obtained from Australian Torresian crows (Corvus orru) in 2019 and 2020. We successfully obtained draft genomes for three avian C. abortus strains (C1, C2 and C3), each comprising draft chromosomes with lengths of 1 115 667, 1 120 231 and 1 082 115 bp, and associated 7 553 bp plasmids, with a genome completeness exceeding 92 %. Molecular characterization revealed that these three strains comprise a novel sequence type (ST333), whilst phylogenetic analyses placed all three strains in a cluster with other avian C. abortus genomes. Interestingly, these three strains share a distant genomic relation (2693 single nucleotide variants) with the reference strain 15-58d/44 (ST152), isolated from a Eurasian magpie (Pica pica) in Poland, highlighting the need for more publicly available genomes. Broad comparative analyses with other avian C. abortus genomes revealed that the three draft genomes contain conserved Chlamydia genomic features, including genes coding for type III secretion system and polymorphic membrane proteins, and potential virulence factors such as the large chlamydial cytotoxin, warranting further studies. This research provides the first avian C. abortus draft genomes from Australian birds, highlighting Torresian crows as novel reservoir hosts for these potential pathogens, and demonstrates a practical methodology for sequencing novel Chlamydia genomes without relying on traditional cell culture.

Characterization of translocon proteins in the type III secretion system of Lawsonia intracellularis

Lawsonia intracellularis, the etiologic agent of proliferative enteropathy (PE), is an obligate intracellular Gram-negative bacterium possessing a type III secretion system (T3SS), which enables the pathogen to translocate effector proteins into targeted host cells to modulate their functions. T3SS is a syringe-like apparatus consisting of a base, an extracellular needle, a tip, and a translocon. The translocon proteins assembled by two hydrophobic membrane proteins can form pores in the host-cell membrane, and therefore play an essential role in the function of T3SS. To date, little is known about the T3SS and translocon proteins of L. intracellularis. In this study, we first analyzed the conservation of the T3S apparatus between L. intracellularis and Yersinia, and characterized the putative T3S hydrophobic major translocon protein LI1158 and minor translocon protein LI1159 in the L. intracellularis genome. Then, by using Yersinia pseudotuberculosis as a surrogate system, we found that the full-length LI1158 and LI1159 proteins, but not the putative class II chaperone LI1157, were secreted in a − Ca2+ and T3SS-dependent manner and the secretion signal was located at the N terminus (aa 1–40). Furthermore, yeast-two hybrid experiments revealed that LI1158 and LI1159 could self-interact, and LI1159 could interact with LI1157. However, unlike CPn0809 and YopB, which are the major hydrophobic translocon proteins of the T3SS of C. pneumoniae and Yersinia, respectively, full-length LI1158 was non-toxic to both yeast and Escherichia coli cells, but full-length LI1159 showed certain toxicity to E. coli cells. Taken together, despite some differences from the findings in other bacteria, our results demonstrate that LI1158 and LI1159 may be the translocon proteins of L. intracellularis T3SS, and probably play important roles in the translocation of effector proteins at the early pathogen infection stage.

Evasion of host antioxidative response via disruption of NRF2 signaling in fatal Ehrlichia-induced liver injury.

Ehrlichia is Gram negative obligate intracellular bacterium that cause human monocytotropic ehrlichiosis (HME). HME is characterized by acute liver damage and inflammation that may progress to fatal toxic shock. We previously showed that fatal ehrlichiosis is due to deleterious activation of inflammasome pathways, which causes excessive inflammation and liver injury. Mammalian cells have developed mechanisms to control oxidative stress via regulation of nuclear factor erythroid 2 related 2 (NRF2) signaling. However, the contribution of NRF2 signaling to Ehrlichia-induced inflammasome activation and liver damage remains elusive. In this study, we investigated the contribution of NRF2 signaling in hepatocytes (HCs) to the pathogenesis of Ehrlichia-induced liver injury following infection with virulent Ixodes ovatus Ehrlichia (IOE, AKA E. japonica). Employing murine model of fatal ehrlichiosis, we found that virulent IOE inhibited NRF2 signaling in liver tissue of infected mice and in HCs as evidenced by downregulation of NRF2 expression, and downstream target GPX4, as well as decreased NRF2 nuclear translocation, a key step in NRF2 activation. This was associated with activation of non-canonical inflammasomes pathway marked by activation of caspase 11, accumulation of reactive oxygen species (ROS), mitochondrial dysfunction, and endoplasmic reticulum (ER) stress. Mechanistically, treatment of IOE-infected HCs with the antioxidant 3H-1,2-Dithiole-3-Thione (D3T), that induces NRF2 activation, attenuated oxidative stress and caspase 11 activation, as well as restored cell viability. Importantly, treatment of IOE-infected mice with D3T resulted in attenuated liver pathology, decreased inflammation, enhanced bacterial clearance, prolonged survival, and resistance to fatal ehrlichiosis. Our study reveals, for the first time, that targeting anti-oxidative signaling pathway is a key approach in the treatment of severe and potential Ehrlichia-induced acute liver injury and sepsis.

Hepatocyte-specific regulation of autophagy and inflammasome activation via MyD88 during lethal Ehrlichia infection.

Hepatocytes play a crucial role in host response to infection. Ehrlichia is an obligate intracellular bacterium that causes potentially life-threatening human monocytic ehrlichiosis (HME) characterized by an initial liver injury followed by sepsis and multi-organ failure. We previously showed that infection with highly virulent Ehrlichia japonica (E. japonica) induces liver damage and fatal ehrlichiosis in mice via deleterious MyD88-dependent activation of CASP11 and inhibition of autophagy in macrophage. While macrophages are major target cells for Ehrlichia, the role of hepatocytes (HCs) in ehrlichiosis remains unclear. We investigated here the role of MyD88 signaling in HCs during infection with E. japonica using primary cells from wild-type (WT) and MyD88-/- mice, along with pharmacologic inhibitors of MyD88 in a murine HC cell line. Similar to macrophages, MyD88 signaling in infected HCs led to deleterious CASP11 activation, cleavage of Gasdermin D, secretion of high mobility group box 1, IL-6 production, and inflammatory cell death, while controlling bacterial replication. Unlike macrophages, MyD88 signaling in Ehrlichia-infected HCs attenuated CASP1 activation but activated CASP3. Mechanistically, active CASP1/canonical inflammasome pathway negatively regulated the activation of CASP3 in infected MyD88-/- HCs. Further, MyD88 promoted autophagy induction in HCs, which was surprisingly associated with the activation of the mammalian target of rapamycin complex 1 (mTORC1), a known negative regulator of autophagy. Pharmacologic blocking mTORC1 activation in E. japonica-infected WT, but not infected MyD88-/- HCs, resulted in significant induction of autophagy, suggesting that MyD88 promotes autophagy during Ehrlichia infection not only in an mTORC1-indpenedent manner, but also abrogates mTORC1-mediated inhibition of autophagy in HCs. In conclusion, this study demonstrates that hepatocyte-specific regulation of autophagy and inflammasome pathway via MyD88 is distinct than MyD88 signaling in macrophages during fatal ehrlichiosis. Understanding hepatocyte-specific signaling is critical for the development of new therapeutics against liver-targeting pathogens such as Ehrlichia.

Clinical and laboratory features of rickettsioses in Yucatan, Mexico

Rickettsia species are obligate intracellular bacteria that can cause mild to severe human disease. Based on phylogeny, clinical symptoms, and antigenic properties, rickettsiae are classified into four groups. Infections by these agents are characterized by clinical symptoms ranging from self-limited to severe and even fatal febrile illnesses, depending on the Rickettsia spp. involved, the patient's predisposition, and timely medical care.The present study aimed to characterize rickettsial diseases in Yucatan according to clinical and laboratory features appearing in medical records corresponding to 427 samples taken between 2015 and 2018.A study was conducted over the period 2015–2018 on 427 samples. Clinical and laboratory features were documented from the patients' medical records. For molecular diagnosis, blood was collected in 3.8 % sodium citrate as anticoagulant, and DNA was extracted. Single-step and nested PCR amplification was performed using genus-specific primers for the rickettsial 17kDa and ompB genes. The amplicons obtained were purified and sequenced.A total of 22.7 % (97/427) positive cases of Rickettsia spp. were identified by PCR from 14.15 % (15/106) of the municipalities in Yucatan. 75.2 % (73/97) of the cases were from the city of Merida during the autumn (September-December). The age groups with the highest frequency of confirmed cases were pediatric (5–14 years) (57.7 %) and adults (25-49 years) (42.2 %). There were six fatal cases in children, one associated with R. typhi and five with R. rickettsii. In non-fatal cases, 32.9 % (32/97) corresponded to the spotted fever group (SFG), and 60.8 % (59/97) to the typhus group (TG). Significant differences in signs, and laboratory data, were observed between the pediatric and adult populations. For the treatment of patients, oral and intravenous doxycycline was used in severe hospitalized cases.Typhus group and spotted fever group Rickettsiae are endemic pathological agents found in urban and rural areas of our region. Molecular identification allows for greater diagnostic accuracy and timely treatment and consequently a better prognosis. It is necessary to implement or reinforce measures focused on the dissemination of knowledge regarding rickettsial diseases and their prevention.

Strains of Anaplasma phagocytophilum from horses in Ohio are related to isolates from humans in the northeastern USA.

The tick-borne obligatory intracellular bacterium Anaplasma phagocytophilum infects granulocytes of humans as well as domesticated and wild animals, causing a febrile disease collectively called granulocytic anaplasmosis. The host species specificity and zoonotic potential of A. phagocytophilum strains remain unclear. In the USA, human granulocytic anaplasmosis (HGA) is primarily reported in Wisconsin and Minnesota, the northeastern coastal states, and northern California. In horses, A. phagocytophilum causes equine granulocytic anaplasmosis (EGA). Although EGA has been reported in the same US regions as HGA, horses with a definitive diagnosis of EGA have not been reported in Ohio, where the incidence of HGA is low. In this study, samples from archived blood smears or whole blood from three horses from three different counties in Ohio that presented in 2018, 2020, and 2021 with clinical signs and laboratory findings consistent with EGA were evaluated by PCR and sequencing. Sequences of ankA (encoding ankyrin A) of the three A. phagocytophilum strains were identical and related to strains isolated from humans in the northeastern USA yet distinct from those in California or Europe based on phylogenetic analysis. Each Ohio strain contains unique A. phagocytophilum p44 genes encoding the immunodominant major outer membrane proteins, underscoring the antigenic diversity of A. phagocytophilum strains that cause EGA in Ohio. From 2018 to 2023, 14 additional cases of EGA were identified by serodiagnosis in Ohio and nearby locations in Pennsylvania, pointing to a heretofore unrecognized prevalence of EGA and the risk of transmission of anaplasmosis to humans and animals in this region.IMPORTANCEThe tick-borne obligatory intracellular bacterium Anaplasma phagocytophilum infects humans as well as domesticated and wild animals, causing a febrile disease collectively called granulocytic anaplasmosis. The epidemiology and the host species specificity and zoonotic potential of A. phagocytophilum strains remain unclear. In this study, ankA (encoding ankyrin A) and p44 gene sequences of A. phagocytophilum were determined in clinical specimens from horses in Ohio and compared with those found in A. phagocytophilum strains from various hosts and geographic regions. With increasing numbers of seropositive horses, the study points out the unrecognized prevalence and uncharacterized strains of A. phagocytophilum infection in horses and the importance of A. phagocytophilum molecular testing for the prevention of equine and human granulocytic anaplasmosis.

Orientia tsutsugamushi: comprehensive analysis of the mobilome of a highly fragmented and repetitive genome reveals the capacity for ongoing lateral gene transfer in an obligate intracellular bacterium.

Obligate intracellular bacteria, or those only capable of growth inside other living cells, have limited opportunities for horizontal gene transfer with other microbes due to their isolated replicative niche. The human pathogen Ot, an obligate intracellular bacterium causing scrub typhus, encodes an unusually high copy number of a ~40 gene mobile genetic element that typically facilitates genetic transfer across microbes. This proliferated element is heavily degraded in Ot and previously assumed to be inactive. Here, we conducted a detailed analysis of this element in eight Ot strains and discovered two strains with at least one intact copy. This implies that the element is still capable of moving across Ot populations and suggests that the genome of this bacterium may be even more dynamic than previously appreciated. Our work raises questions about intracellular microbial evolution and sounds an alarm for gene-based efforts focused on diagnosing and combatting scrub typhus.

Plasticity in the cell division processes of obligate intracellular bacteria.

Most bacteria divide through a highly conserved process called binary fission, in which there is symmetric growth of daughter cells and the synthesis of peptidoglycan at the mid-cell to enable cytokinesis. During this process, the parental cell replicates its chromosomal DNA and segregates replicated chromosomes into the daughter cells. The mechanisms that regulate binary fission have been extensively studied in several model organisms, including Eschericia coli, Bacillus subtilis, and Caulobacter crescentus. These analyses have revealed that a multi-protein complex called the divisome forms at the mid-cell to enable peptidoglycan synthesis and septation during division. In addition, rod-shaped bacteria form a multi-protein complex called the elongasome that drives sidewall peptidoglycan synthesis necessary for the maintenance of rod shape and the lengthening of the cell prior to division. In adapting to their intracellular niche, the obligate intracellular bacteria discussed here have eliminated one to several of the divisome gene products essential for binary fission in E. coli. In addition, genes that encode components of the elongasome, which were mostly lost as rod-shaped bacteria evolved into coccoid organisms, have been retained during the reductive evolutionary process that some coccoid obligate intracellular bacteria have undergone. Although the precise molecular mechanisms that regulate the division of obligate intracellular bacteria remain undefined, the studies summarized here indicate that obligate intracellular bacteria exhibit remarkable plasticity in their cell division processes.

Granulocytic anaplasmosis in cats from central Europe and molecular characterization of feline Anaplasma phagocytophilum strains by ankA gene, groEL gene and multilocus sequence typing

BackgroundAnaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It is transmitted by ticks of the Ixodes ricinus complex and causes febrile illness called granulocytic anaplasmosis primarily in humans, horses, dogs, sheep, cattle and goats. In comparison, clinically apparent disease has been described rarely in cats especially compared to dogs and horses. It is currently unknown whether cats are less susceptible to A. phagocytophilum or whether granulocytic anaplasmosis might be underdiagnosed in cats.MethodsTo address this question, we examined clinical signs and laboratory findings in seven A. phagocytophilum infected cats from Germany and Switzerland. We then genetically characterized feline A. phagocytophilum strains and compared them to those from other hosts showing clinically apparent disease. For this purpose, ankA-based, groEL-based and multilocus sequence typing (MLST) were applied. Furthermore, the concordance between these typing methods was assessed.ResultsFever, lethargy and anorexia were the most common clinical signs in cats suffering from granulocytic anaplasmosis. The most frequent laboratory finding was thrombocytopenia. All three typing methods consistently indicated that the A. phagocytophilum strains found infecting cats are the same as those that cause disease in humans, dogs and horses. In general, the three typing methods applied exhibited high concordance.ConclusionsThe genetic characterization of the feline A. phagocytophilum strains indicates that strain divergence is not the explanation for the fact that granulocytic anaplasmosis is much less frequently diagnosed in cats than in dogs and horses. Otherwise, it may be possible that cats are less susceptible to the same strains than dogs and horse are. However, due to the unspecific clinical signs, it should be considered that granulocytic anaplasmosis may be under-diagnosed in cats.Graphical

Identification of the alternative sigma factor regulons of Chlamydia trachomatis using multiplexed CRISPR interference.

Chlamydia trachomatis is a developmentally regulated, obligate intracellular bacterium that encodes three sigma factors: σ66, σ54, and σ28. σ66 is the major sigma factor controlling most transcription initiation during early- and mid-cycle development as the infectious elementary body (EB) transitions to the non-infectious reticulate body (RB) that replicates within an inclusion inside the cell. The roles of the minor sigma factors, σ54 and σ28, have not been well characterized to date; however, there are data to suggest each functions in late-stage development and secondary differentiation as RBs transition to EBs. As the process of secondary differentiation itself is poorly characterized, clarifying the function of these alternative sigma factors by identifying the genes regulated by them will further our understanding of chlamydial differentiation. We hypothesize that σ54 and σ28 have non-redundant and essential functions for initiating late gene transcription thus mediating secondary differentiation in Chlamydia. Here, we demonstrate the necessity of each minor sigma factor in successfully completing the developmental cycle. We have implemented and validated multiplexed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference techniques, novel to the chlamydial field to examine the effects of knocking down each alternative sigma factor individually and simultaneously. In parallel, we also overexpressed each sigma factor. Altering transcript levels for either or both alternative sigma factors resulted in a severe defect in EB production as compared to controls. Furthermore, RNA sequencing identified differentially expressed genes during alternative sigma factor dysregulation, indicating the putative regulons of each. These data demonstrate that the levels of alternative sigma factors must be carefully regulated to facilitate chlamydial growth and differentiation. IMPORTANCE Chlamydia trachomatis is a significant human pathogen in both developed and developing nations. Due to the organism's unique developmental cycle and intracellular niche, basic research has been slow and arduous. However, recent advances in chlamydial genetics have allowed the field to make significant progress in experimentally interrogating the basic physiology of Chlamydia. Broadly speaking, the driving factors of chlamydial development are poorly understood, particularly regarding how the later stages of development are regulated. Here, we employ a novel genetic tool for use in Chlamydia while investigating the effects of dysregulating the two alternative sigma factors in the organism that help control transcription initiation. We provide further evidence for both sigma factors' essential roles in late-stage development and their potential regulons, laying the foundation for deeper experimentation to uncover the molecular pathways involved in chlamydial differentiation.

Diagnosis and Management of Rickettsial Infections among Febrile Patients Attending in Inpatients and Outpatient Department in a District Hospital in Bangladesh

Background: Numerous obligatory intracellular bacteria from the Alphaproteobacteria genus Rickettsia can cause rickettsial infection. The most prevalent newly emerging and reemerging diseases are rickettsial infections. Objectives: The aim of the study was diagnosis and management of rickettsial infections among febrile patients attending in inpatient and outpatient department in a district hospital in Bangladesh. Methods: This cross-section observational study was carried out in the 250 Beded District Hospital, Joypurhat. The duration of the period from March 2022 to February 2023. A total of 250 patients participated in the study. Both male and female with any age, patients with elevated body temperature, headache, malaise, rash, nausea, and vomiting and gave consent to be included in the study. Severely ill patients, not willing to participate were excluded from the study. Weil Felix test was used in this study and a titre of 1:80 was considered to be positive. Statistical evaluation of the results used to be obtained via the use of a window-based computer software program devised with Statistical Packages for Social Sciences (SPSS-24). Results: The mean age of the patients was 39 ± 1.9. 8% of the patients had ≤18 years of age. 16% were within the age group of 19-29 years, 28% were within the age group of 30-39 years, 24% were within the age group of 40-49 years, 14.4% were within the age group of 50-59 years and 9.6% were ≥60 years and 52% of the patients were male. 100% of the respondents had fever, 20% had generalized weakness, 15.2% had headache, 16% had cough and cold, 24% had body ache, 8% had vomiting and abdominal pain respectively, 4% had per nasal watery discharge, 36% had rash, 16% had nausea, 24% had anorexia and 4% had other symptoms. CBC & ESR and Weil Felix test were the most common (100%) investigation among the patients, Urine R/M/E in 68%, serum creatinine in 18%, USG of W/A in 15%, CRP in 18%, PBF in 7, Urine C/S in 10%, S. Electrolytes in 4% cases. We found 90 Rickettsial Infection positive patients. Doxycycline and Paracetamol and Esomeprazole were prescribed to all patients 90(100%) and followed by Linagliptin+Metformin to 20(22.22%), Glimipiride to 19(21.11%), Azithromycin to 18(20%) and Domperidon to 10(1.11%), Montelukast 9 (10%), Pantoprazole to 8(8.89%), Rupatadine to 7(7.77%), Ondansetron to 6(6.67%), Tiemonium to 5 5.57%), Fexofenadin to4(4.44%) and other treatment along with the prescribed medicines was given to 40(44.44%) patients. Authors studied the response of Doxycycline which showed 85(94.44%) cases improved within 48 hours of start of treatment. Remaining 5(5.55%) cases showed delayed response due to late presentation. Conclusion: Rickettsial illness is inextricably linked to the sociocultural life of the majority of metropolitan residents. However, a lack of medical facilities and qualified doctors makes it challenging to identify rickettsial disease in febrile patients. It's important to make an early diagnosis when treating rickettsial illness.

Hidden from plain sight: Novel Simkaniaceae and Rhabdochlamydiaceae diversity emerging from screening genomic and metagenomic data

Chlamydiota are an ancient and hyperdiverse phylum of obligate intracellular bacteria. The best characterized representatives are pathogens or parasites of mammals, but it is thought that their most common hosts are microeukaryotes like Amoebozoa. The diversity in taxonomy, evolution, and function of non-pathogenic Chlamydiota are slowly being described. Here we use data mining techniques and genomic analysis to extend our current knowledge of Chlamydiota diversity and its hosts, in particular the Order Parachlamydiales. We extract one Rhabdochlamydiaceae and three Simkaniaceae Metagenome-Assembled Genomes (MAGs) from NCBI Short Read Archive deposits of ciliate and algal genome sequencing projects. We then use these to identify a further 14 and 8 MAGs respectively amongst existing, unidentified environmental assemblies. From these data we identify two novel clades with host associated data, for which we propose the names “Sacchlamyda saccharinae” (Family Rhabdochlamydiaceae) and “Amphrikana amoebophyrae” (Family Simkaniaceae), as well as a third new clade of environmental MAGs “Acheromyda pituitae” (Family Rhabdochlamydiaceae). The extent of uncharacterized diversity within the Rhabdochlamydiaceae and Simkaniaceae is indicated by 16 of the 22 MAGs being evolutionarily distant from currently characterised genera. Within our limited data, there was great predicted diversity in Parachlamydiales metabolism and evolution, including the potential for metabolic and defensive symbioses as well as pathogenicity. These data provide an imperative to link genomic diversity in metagenomics data to their associated eukaryotic host, and to develop onward understanding of the functional significance of symbiosis with this hyperdiverse clade.

First isolation of Rickettsia amblyommatis from Amblyomma mixtum in Colombia

BackgroundRickettsiae are obligate intracellular Gram-negative bacteria that are the causative agent of rickettsioses and are spread to vertebrate hosts by arthropods. There are no previous reports of isolation of Rickettsia amblyommatis for Colombia.MethodsA convenience sampling was executed in three departments in Colombia for direct collection of adult ticks on domestic animals or over vegetation. Ticks were screened for the presence of Rickettsia spp. by real-time polymerase chain reaction (qPCR) amplifying the citrate synthase gene (gltA), and the positive sample was processed for isolation and further molecular characterization by conventional PCR. The absolute and relative frequencies were calculated for several tick species variables. All products from conventional PCR were further purified and sequenced by the Sanger technique. Representative sequences of 18 Rickettsia species were downloaded from GenBank. Consensus phylogenetic trees were constructed for the gltA, ompB, ompA, and htrA genes with 1000 replicates, calculating bootstrap values through the maximum likelihood method and the generalized time reversible substitution model in the MEGA 7.0 software program.ResultsOne female Amblyomma mixtum collected on vegetation was amplified by qPCR (gltA), indicating a frequency of 1.6% (1/61) for Rickettsia spp. infection. Sequence analysis of a rickettsial isolate from this tick in BLASTn showed 100% identity with gltA (340 base pairs [bp]), 99.87% for ompB (782 bp), 98.99% for htrA (497 bp), and 100% for ompA (488 bp) to R. amblyommatis. Concatenated phylogenetic analysis confirmed these findings indicating that the isolate is grouped with other sequences of Amblyomma cajennense complex from Panama and Brazil within the R. amblyommatis clade.ConclusionsThis paper describes the isolation and early molecular identification of a R. amblyommatis strain from A. mixtum in Colombia.Graphical

Serological profile of patients suspected with non-scrub typhus rickettsioses

BackgroundRickettsial pathogens are Gram-negative, obligate intracellular bacteria. They are transmitted by arthropods and are responsible for a wide variety of disease, from minor to life-threatening, which have a global effect on human health. Limited data are available on the prevalence of rickettsial diseases from India, and the disease epidemiology is not fully described. This study aimed to diagnose non-scrub typhus rickettsioses including spotted fever and typhus group of Rickettsia in clinically suspected patients by using standard serological tests and recognition of common epidemiological conditions and clinical manifestations. MethodsDuring the study period, a total of 700 patients of all ages with acute febrile illness were enrolled. Patients were screened for rickettsial infection using IgM Enzyme-linked immunosorbent assay (ELISA) and Immunofluorescence assay (IFA) was performed to confirm the ELISA positive results. The relevant demographic, clinical, and laboratory details of patients were documented and analyzed. ResultsOf 700 samples tested, 141 (20.2%) were found to be positive for IgM antibodies against rickettsioses using ELISA and IFA. SFGR was positive in 15 (2.2%), TGR was positive in 112 (16%) and 14 (2%) samples were positive for both groups. 20 (14.2%) patients required admission to the intensive care unit (ICU), and 24 (17%) in-hospital deaths occurred. ConclusionsThe prevalence of rickettsioses in India appears to be underestimated; therefore, increased awareness and improved diagnostic testing could facilitate early detection of cases, pathogen-targeted appropriate treatment, and improved outcomes for patients. Despite the fact that Rickettsiae can be isolated or detected using molecular techniques in clinical specimens, serology still remains the most commonly used diagnostic method for rickettsioses around the world. Our study helps bridge the gap of limited data on Rickettsia in north India and could be useful for future epidemiological investigation of rickettsial diseases and outbreaks.

Impact of azithromycin, doxycycline and redox-active small molecules on amoxicillin-induced Chlamydia pneumoniae persistence

Amoxicillin is recommended as primary treatment for community-acquired bacterial pneumonia (CABP). 5–10% of CABP cases are caused by Chlamydia pneumoniae, an obligate intracellular bacterium which responds to beta-lactam antibiotics by converting to a persistent phenotype. To support rational pharmacotherapy of C. pneumoniae infections, we investigated how clinically relevant concentrations of azithromycin and doxycycline affect amoxicillin induced C. pneumoniae persistence. Given the known role of redox state alterations in the action of bactericidal antibiotics and widespread use of redox-active dietary supplements when experiencing respiratory symptoms, we also studied how redox active compounds affect the studied antibiotic treatments. Our data demonstrate that clinically applied amoxicillin concentrations (10 and 25 mg/l) fail to eradicate C. pneumoniae infection in respiratory epithelial cells. Transmission electron microscopy (TEM) of amoxicillin-treated C. pneumoniae infected cells reveal aberrant bacterial morphology characteristic of chlamydial stress response. Amoxicillin was also found to significantly limit the antichlamydial effect of azithromycin or doxycycline. However, based on quantitative culture and quantitative PCR data, azithromycin was superior to doxycycline in C. pneumoniae eradication either as monotherapy or in combination with amoxicillin. Amoxicillin was also found to decrease respiratory epithelial cell glutathione (GSH) levels, whereas redox-active dibenzocyclooctadiene lignans increased C. pneumoniae load in amoxicillin-treated cultures up to two-fold. These data highlight the impact of relative administration time on the efficacy of antichlamydial antibiotics and indicate unfavorable interactions between amoxicillin and redox-active small molecules.

Pathogenic Rickettsia, Anaplasma, and Ehrlichia in Rhipicephalus microplus ticks collected from cattle and laboratory hatched tick larvae.

The order Rickettsiales contains a group of vector-borne gram-negative obligate intracellular bacteria, which often cause human emerging infectious diseases and economic losses for dairy and meat industries. The purpose of this study is to investigate the distribution of the pathogens including Rickettsia spp., Anaplasma spp., and Ehrlichia spp. in the order Rickettsiales in ticks from Yueyang, a prefecture-level city of Hunan Province in Sothern China, and assess the potentiality of transovarial transmission of these rickettsial organisms. Ticks were collected from cattle in a farm in Yueyang City and the tick DNA was used as template to amplify the htrA, rrs, gltA, ompA and ompB genes of Rickettsia as well as rrs and groEL genes of Anaplasma and Ehrlichia. All ticks (465) collected were the cattle tick, Rhipicephalus microplus. PCR showed the minimum infection rate (MIR) was 1.5% (7/465) for Candidatus Rickettsia xinyangensis, 1.9% (9/465) for C. Anaplasma boleense, 1.3% (6/465) for Anaplasma platys, 0.6% (3/465) for A. marginale, and 1.17% (2/465) for each of A. bovis, Ehrlichia minasensis, and a non-classified Ehrlichia sp. A human pathogen, C. Rickettsia xinyangensis and A. platys were detected in 100% (3/3) and 33.3% (2/6) laboratory-hatched larval pools from infected females respectively. Our study revealed a diversity of pathogenic rickettsial species in R. microplus ticks from Hunan Province suggesting a threat to people and animals in China. This study also provided the first molecular evidence for the potential transovarial transmission of C. Rickettsia xinyangensis and A. platys in R. microplus, indicating that R. microplus may act as the host of these two pathogens.

LA PREDICCIÓN IN SILICO DETERMINA LA PRESENCIA DE EPÍTOPOS INMUNOGÉNICOS ALTAMENTE ESPECÍFICOS EN FRAGMENTOS DE LA PROTEÍNA DE LA MEMBRANA POLIMÓRFICA (PMP17G) DE Chlamydia abortus

<p><strong>Background.</strong> Ovine Enzootic Abortion is a contagious infectious disease caused by a Gram negative and obligate intracellular bacterium, <em>Chlamydia abortus</em>. For field diagnosis, commercial serological tests are used; however, some of these tests show low sensitivity and specificity rates, due to the cross-reactions that the antigens used have against other pathogens. For the most accurate diagnosis, it is necessary to develop tests with more specific antigens such as polymorphic membrane proteins (Pmp's), that allow to determine the presence of specific epitopes using new technologies. <strong>Objective.</strong> To determine <em>in silico</em> the presence of epitopes with specific immunogenic potential against <em>Chlamydia abortus</em> of two fragments of the PMP17G protein. <strong>Methodology.</strong> The cloning and sequencing of the fragments was carried out from a field isolate of <em>Chlamydia abortus</em>, and from the analysis of these sequences, with the help of two bioinformatics software’s. <strong>Results.</strong> Several epitopes from <em>Chlamydia abortus</em> were found, rPOMP90-3 (eight epitopes) and rPOMP90-4 (one epitope). <strong>Implications.</strong> Bioinformatics analysis indicated that both fragments of the protein have the capacity to activate the immune system, which would be useful for the development of diagnostic kits and immunogens. <strong>Conclusions.</strong> The <em>in silico</em> analysis allowed to efficiently predict and identify specific epitopes against <em>Chlamydia abortus</em> in both fragments of the protein. </p>

The Chlamydia effector CpoS modulates the inclusion microenvironment and restricts the interferon response by acting on Rab35.

The obligate intracellular bacterium Chlamydia trachomatis inserts a family of inclusion membrane (Inc) proteins into the membrane of its vacuole (the inclusion). The Inc CpoS is a critical suppressor of host cellular immune surveillance, but the underlying mechanism remained elusive. By complementing a cpoS mutant with various natural orthologs and variants of CpoS, we linked distinct molecular interactions of CpoS to distinct functions. Unexpectedly, we found CpoS to be essential for the formation of inclusion membrane microdomains that control the spatial organization of multiple Incs involved in signaling and modulation of the host cellular cytoskeleton. While the function of CpoS in microdomains was uncoupled from its role in the suppression of host cellular defenses, we found the ability of CpoS to interact with Rab GTPases to be required not only for the manipulation of membrane trafficking, such as to mediate transport of ceramide-derived lipids (sphingolipids) to the inclusion, but also for the inhibition of Stimulator of interferon genes (STING)-dependent type I interferon responses. Indeed, depletion of Rab35 phenocopied the exacerbated interferon responses observed during infection with CpoS-deficient mutants. Overall, our findings highlight the role of Inc-Inc interactions in shaping the inclusion microenvironment and the modulation of membrane trafficking as a pathogenic immune evasion strategy. IMPORTANCE Chlamydia trachomatis is a prevalent bacterial pathogen that causes blinding ocular scarring and urogenital infections that can lead to infertility and pregnancy complications. Because Chlamydia can only grow within its host cell, boosting the intrinsic defenses of human cells may represent a novel strategy to fight pathogen replication and survival. Hence, CpoS, a Chlamydia protein known to block host cellular defenses, or processes regulated by CpoS, could provide new opportunities for therapeutic intervention. By revealing CpoS as a multifunctional virulence factor and by linking its ability to block host cellular immune signaling to the modulation of membrane trafficking, the present work may provide a foundation for such rationale targeting and advances our understanding of how intracellular bacteria can shape and protect their growth niche.

The Fish Pathogen "Candidatus Clavichlamydia salmonicola"-A Missing Link in the Evolution of Chlamydial Pathogens of Humans.

Chlamydiae like Chlamydia trachomatis and Chlamydia psittaci are well-known human and animal pathogens. Yet, the chlamydiae are a much larger group of evolutionary ancient obligate intracellular bacteria that includes predominantly symbionts of protists and diverse animals. This makes them ideal model organisms to study evolutionary transitions from symbionts in microbial eukaryotes to pathogens of humans. To this end, comparative genome analysis has served as an important tool. Genome sequence data for many chlamydial lineages are, however, still lacking, hampering our understanding of their evolutionary history. Here, we determined the first high-quality draft genome sequence of the fish pathogen "Candidatus Clavichlamydia salmonicola", representing a separate genus within the human and animal pathogenic Chlamydiaceae. The "Ca. Clavichlamydia salmonicola" genome harbors genes that so far have been exclusively found in Chlamydia species suggesting that basic mechanisms important for the interaction with chordate hosts have evolved stepwise in the history of chlamydiae. Thus, the genome sequence of "Ca. Clavichlamydia salmonicola" allows to constrain candidate genes to further understand the evolution of chlamydial virulence mechanisms required to infect mammals.