Impact of Three Different Mutations in Ehrlichia chaffeensis in Altering the Global Gene Expression Patterns

Citation: Kondethimmanahalli, C., & Ganta, R. (2018). Impact of Three Different Mutations in Ehrlichia chaffeensis in Altering the Global Gene Expression Patterns. Scientific Reports, 8(1). https://doi.org/10.1038/s41598-018-24471-3The rickettsial pathogen Ehrlichia chaffeensis causes a tick-borne disease, human monocytic ehrlichiosis. Mutations within certain genomic locations of the pathogen aid in understanding the pathogenesis and in developing attenuated vaccines. Our previous studies demonstrated that mutations in different genomic sites in E. chaffeensis caused variable impacts on their growth and attenuation in vertebrate and tick hosts. Here, we assessed the effect of three mutations on transcriptional changes using RNA deep-sequencing technology. RNA sequencing aided in detecting 66–80% of the transcripts of wildtype and mutant E. chaffeensis. Mutation in an antiporter gene (ECH_0379) causing attenuated growth in vertebrate hosts resulted in the down regulation of many transcribed genes. Similarly, a mutation downstream to the ECH_0490 coding sequence resulted in minimal impact on the pathogen’s in vivo growth, but caused major changes in its transcriptome. This mutation caused enhanced expression of several host stress response genes. Even though the ECH_0660 gene mutation caused the pathogen’s rapid clearance in vertebrate hosts and aids in generating a protective response, there was minimal impact on the transcriptome. The transcriptomic data offer novel insights about the impact of mutations on global gene expression and how they may contribute to the pathogen’s resistance and/or clearance from the host

First trimester uric acid level: a reliable marker for gestational diabetes mellitus

Background: The prevalence of diabetes mellitus (DM) is increasing worldwide and more in developing countries like India. The diabetic epidemic experienced in India can be due to strong genetic factors coupled with increasing urbanization, sedentary lifestyle, changes in the dietary patterns and increasing obesity. Indians are at an 11-fold increased risk of developing gestational glucose intolerance and hence universal screening is essential. Uric acid is a known marker of oxidative stress. Hyperuricemia in early pregnancy may be an indicator of the existing metabolic disturbance which can hinder the maternal physiological adaptations generally seen in pregnancy thus making the pregnant women more vulnerable to the development of gestational diabetes mellitus. The objective of this study was to investigate the association between elevated uric acid levels in the first trimester of pregnancy with gestational diabetes.Methods: This prospective observational study was conducted in Chinmaya mission hospital, Bangalore from June 2016 to March 2017 (10 months). Three hundred and twelve (312) pregnant women of gestational age less than 12 weeks who attended the OBG outpatient department within this time of period for regular antenatal check-up were enrolled in the study. Along with the other antenatal investigations serum uric acid levels were estimated before 12 weeks and also between 24-28 weeks. At 24-28 weeks screening for GDM was done by OGCT using 75 gms of glucose (IADPISG criteria). Other parameters like age, parity, BMI, family history of diabetes was noted and compared.Results: In our study, among the 312 pregnant women, 88 (28%) developed GDM. Of these 74 Women (84%) with GDM had uric acid levels above 3.5 mg/dl and 14 women (15.9%) with GDM had uric acid levels below 3.5 mg/dl. Women with higher BMI showed high uric acid levels.Conclusions: Elevated serum uric acid in the first trimester has a significant correlation with development of GDM. In present study; the cut-off level of maternal serum uric acid of 3.5 mg/dl in the first trimester appears to have a good sensitivity and specificity in identifying those patients who are most likely to develop GDM later in pregnancy

Promoter analysis of macrophage- and tick cell-specific differentially expressed Ehrlichia chaffeensis p28-Omp genes

<p>Abstract</p> <p>Background</p> <p><it>Ehrlichia chaffeensis </it>is a rickettsial agent responsible for an emerging tick-borne illness, human monocytic ehrlichiosis. Recently, we reported that <it>E. chaffeensis </it>protein expression is influenced by macrophage and tick cell environments. We also demonstrated that host response differs considerably for macrophage and tick cell-derived bacteria with delayed clearance of the pathogen originating from tick cells.</p> <p>Results</p> <p>In this study, we mapped differences in the promoter regions of two genes of p28-Omp locus, genes 14 and 19, whose expression is influenced by macrophage and tick cell environments. Primer extension and quantitative RT-PCR analysis were performed to map transcription start sites and to demonstrate that <it>E. chaffeensis </it>regulates transcription in a host cell-specific manner. Promoter regions of genes 14 and 19 were evaluated to map differences in gene expression and to locate RNA polymerase binding sites.</p> <p>Conclusion</p> <p>RNA analysis and promoter deletion analysis aided in identifying differences in transcription, DNA sequences that influenced promoter activity and RNA polymerase binding regions. This is the first description of a transcriptional machinery of <it>E. chaffeensis</it>. In the absence of available genetic manipulation systems, the promoter analysis described in this study can serve as a novel molecular tool for mapping the molecular basis for gene expression differences in <it>E. chaffeensis </it>and other related pathogens belonging to the <it>Anaplasmataceae </it>family.</p

Comparative Experimental Infection Study in Dogs with Ehrlichia canis, E. chaffeensis, Anaplasma platys and A. phagocytophilum

Citation: Nair, A. D. S., Cheng, C., Ganta, C. K., Sanderson, M. W., Alleman, A. R., Munderloh, U. G., & Ganta, R. R. (2016). Comparative Experimental Infection Study in Dogs with Ehrlichia canis, E. chaffeensis, Anaplasma platys and A. phagocytophilum. Plos One, 11(2), 21. doi:10.1371/journal.pone.0148239Dogs acquire infections with the Anaplasmataceae family pathogens, E. canis, E. chaffeensis, E. ewingii, A. platys and A. phagocytophilum mostly during summer months when ticks are actively feeding on animals. These pathogens are also identified as causing diseases in people. Despite the long history of tick-borne diseases in dogs, much remains to be defined pertaining to the clinical and pathological outcomes of infections with these pathogens. In the current study, we performed experimental infections in dogs with E. canis, E. chaffeensis, A. platys and A. phagocytophilum. Animals were monitored for 42 days to evaluate infection-specific clinical, hematological and pathological differences. All four pathogens caused systemic persistent infections detectible throughout the 6 weeks of infection assessment. Fever was frequently detected in animals infected with E. canis, E. chaffeensis, and A. platys, but not in dogs infected with A. phagocytophilum. Hematological differences were evident in all four infected groups, although significant overlap existed between the groups. A marked reduction in packed cell volume that correlated with reduced erythrocytes and hemoglobin was observed only in E. canis infected animals. A decline in platelet numbers was common with E. canis, A. platys and A. phagocytophilum infections. Histopathological lesions in lung, liver and spleen were observed in all four groups of infected dogs; infection with E. canis had the highest pathological scores, followed by E. chaffeensis, then A. platys and A. phagocytophilum. All four pathogens induced IgG responses starting on day 7 post infection, which was predominantly comprised of IgG2 subclass antibodies. This is the first detailed investigation comparing the infection progression and host responses in dogs after inoculation with four pathogens belonging to the Anaplasmataceae family. The study revealed a significant overlap in clinical, hematological and pathological changes resulting from the infections

CPK: The new tool in the diagnosis of ectopic pregnancy

Background: Ectopic pregnancy is still a diagnostic dilemma presenting with various complaints. The classic triad of amenorrhea, abdominal pain, vaginal bleeding and /or syncope is not always seen. Misdiagnosis can lead to delay in treatment, blood loss is found to be the major cause of death. Early and accurate diagnosis is critical in bringing down the maternal mortality and morbidity. Prompt and effective treatment of an ectopic pregnancy can help preserve the chances of future healthy pregnancies. Aim of present study was to investigate whether creatinine phosphokinase (cpk) can be used as an effective diagnostic tool in the early diagnosis of ectopic pregnancy which can help in decreasing the maternal mortality and morbidityMethods: This observational comparative three group clinical study was conducted at Chinmaya Mission Hospital, Bangalore, between May 2016 to January 2017.120 women in their early trimester were studied of which 40 were diagnosed cases of ectopic pregnancies, 40 women presented with intrauterine abortive pregnancies and 40 women had normal healthy pregnancies. Serum CPK, serum B-HCG, vaginal scans were done in all, along with routine investigations.Results: The mean CPK values in normal, abortive and ectopic pregnancies were 36.92±6.44, 43.95±11.96 and 91.55±30.43 respectively. It was found to be significantly higher in ectopic Pregnancies. Also, the mean CPK in ruptured and unruptured ectopic pregnancy were 97.26±25.97 and 63.82±34.92 respectively.Conclusions: Present study shows that maternal CPK levels are significantly higher in women with ectopic pregnancies. CPK can serve as the reliable biochemical marker to diagnose ectopic pregnancy particularly ruptured. CPK can be used to increase the diagnostic efficacy in ectopic pregnancy, which followed by rapid and appropriate treatment can reduce the mortality, morbidity and preserve future fertility

A genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium, Ehrlichia chaffeensis

Citation: Wang, Y., Wei, L., Liu, H., Cheng, C., & Ganta, R. R. (2017). A genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium, Ehrlichia chaffeensis. Scientific Reports, 7(1). https://doi.org/10.1038/s41598-017-16023-yObligate intracellular bacteria (obligates) belonging to Rickettsiales and Chlamydiales cause diseases in hundreds of millions of people worldwide and in many animal species. Lack of an efficient system for targeted mutagenesis in obligates remains a major impediment in understanding microbial pathogenesis. Challenges in creating targeted mutations may be attributed to essential nature of majority of the genes and intracellular replication dependence. Despite success in generating random mutations, a method that works well in creating mutations in specific genes of interest followed by complementation remains problematic for obligates and is a highly sought-after goal. We describe protocols to generate stable targeted mutations by allelic exchange in Ehrlichia chaffeensis, an obligate intracellular tick-borne bacterium responsible for human monocytic ehrlichiosis. Targeted mutations in E. chaffeensis were created to disrupt two genes, and also to restore one gene by another allelic exchange mutation leading to the restoration of transcription and protein expression from the inactivated gene and the recovered organisms also express mCherry, which distinguishes from the wild type. We expect that the methods developed are broadly applicable to other obligates, particularly to rickettsial pathogens, to routinely perform targeted mutations to enable studies focused on protein structure-function analyses, host-pathogen interactions and in developing vaccines

Assessment of a liquid larval diet for rearing Dacus species and Bactrocera dorsalis (Diptera: Tephritidae)

Species of the genus Dacus are important insect pests of fruits and vegetables. Two Dacus species, Dacus punctatifrons Karsch and Dacus vertebratus Bezzi, as well as Bactrocera dorsalis (Hendel), were reared on a liquid artificial diet, a carrot (Daucus carota L.)-based solid artificial diet, and a natural fruit host to assess the suitability of the liquid diet for small-scale rearing of these species. Egg hatch, pupal production, adult emergence and F1 productivity were recorded to evaluate performance of the three species on each diet. Egg hatch on the three diets was more than 50% for D.&nbsp;punctatifrons and B.&nbsp;dorsalis, but for D.&nbsp;vertebratus, egg hatch was less than 40% when they were introduced to the liquid artificial diet. Pupal production for both Dacus species was very low or nil on the liquid artificial diet and the carrot-based artificial diet, respectively. Adult emergence was low for D.&nbsp;punctatifrons and nil for D.&nbsp;vertebratus on the liquid artificial diet. This study showed that the two Dacus species did not develop well on either the liquid or solid carrot-based artificial diet whereas B.&nbsp;dorsalis performed well on the liquid diet. Cucumber, the natural host of both Dacus species, was better for small-scale rearing of these species than the liquid and carrot-based artificial diets. Nutrients found in cucumber need to be identified to formulate alternative rearing media for Dacus species that are economical and easy to use

Transcription of Ehrlichia chaffeensis genes is accomplished by RNA polymerase holoenzyme containing either sigma 32 or sigma 70

Citation: Liu, H., Ohlen, T. V., Cheng, C., Faburay, B., & Ganta, R. R. (2013). Transcription of Ehrlichia chaffeensis Genes Is Accomplished by RNA Polymerase Holoenzyme Containing either Sigma 32 or Sigma 70. PLOS ONE, 8(11), e81780. https://doi.org/10.1371/journal.pone.0081780Bacterial gene transcription is initiated by RNA polymerase containing a sigma factor. To understand gene regulation in Ehrlichia chaffeensis, an important tick-transmitted rickettsiae responsible for human monocytic ehrlichiosis, we initiated studies evaluating the transcriptional machinery of several genes of this organism. We mapped the transcription start sites of 10 genes and evaluated promoters of five genes (groE, dnaK, hup, p28-Omp14 and p28-Omp19 genes). We report here that the RNA polymerase binding elements of E. chaffeensis gene promoters are highly homologous for its only two transcription regulators, sigma 32 and sigma 70, and that gene expression is accomplished by either of the transcription regulators. RNA analysis revealed that although transcripts for both sigma 32 and sigma 70 are upregulated during the early replicative stage, their expression patterns remained similar for the entire replication cycle. We further present evidence demonstrating that the organism’s -35 motifs are essential to transcription initiations. The data suggest that E. chaffeensis gene regulation has evolved to support the organism’s growth, possibly to facilitate its intraphagosomal growth. Considering the limited availability of genetic tools, this study offers a novel alternative in defining gene regulation in E. chaffeensis and other related intracellular pathogens

Mutations in Ehrlichia chaffeensis Causing Polar Effects in Gene Expression and Differential Host Specificities

Citation: Cheng, C. M., Nair, A. D. S., Jaworski, D. C., & Ganta, R. R. (2015). Mutations in Ehrlichia chaffeensis Causing Polar Effects in Gene Expression and Differential Host Specificities. Plos One, 10(7), 13. doi:10.1371/journal.pone.0132657Ehrlichia chaffeensis, a tick-borne rickettsial, is responsible for human monocytic ehrlichiosis. In this study, we assessed E. chaffeensis insertion mutations impacting the transcription of genes near the insertion sites. We presented evidence that the mutations within the E. chaffeensis genome at four genomic locations cause polar effects in altering gene expressions. We also reported mutations causing attenuated growth in deer (the pathogen's reservoir host) and in dog (an incidental host), but not in its tick vector, Amblyomma americanum. This is the first study documenting insertion mutations in E. chaffeensis that cause polar effects in altering gene expression from the genes located upstream and downstream to insertion sites and the differential requirements of functionally active genes of the pathogen for its persistence in vertebrate and tick hosts. This study is important in furthering our knowledge on E. chaffeensis pathogenesis