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

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

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

Ehrlichia chaffeensis Infection in the Reservoir Host (White-Tailed Deer) and in an Incidental Host (Dog) Is Impacted by Its Prior Growth in Macrophage and Tick Cell Environments

Citation: Nair, A. D. S., Cheng, C., Jaworski, D. C., Willard, L. H., Sanderson, M. W., & Ganta, R. R. (2014). Ehrlichia chaffeensis Infection in the Reservoir Host (White-Tailed Deer) and in an Incidental Host (Dog) Is Impacted by Its Prior Growth in Macrophage and Tick Cell Environments. PLOS ONE, 9(10), e109056. https://doi.org/10.1371/journal.pone.0109056Ehrlichia chaffeensis, transmitted from Amblyomma americanum ticks, causes human monocytic ehrlichiosis. It also infects white-tailed deer, dogs and several other vertebrates. Deer are its reservoir hosts, while humans and dogs are incidental hosts. E. chaffeensis protein expression is influenced by its growth in macrophages and tick cells. We report here infection progression in deer or dogs infected intravenously with macrophage- or tick cell-grown E. chaffeensis or by tick transmission in deer. Deer and dogs developed mild fever and persistent rickettsemia; the infection was detected more frequently in the blood of infected animals with macrophage inoculum compared to tick cell inoculum or tick transmission. Tick cell inoculum and tick transmission caused a drop in tick infection acquisition rates compared to infection rates in ticks fed on deer receiving macrophage inoculum. Independent of deer or dogs, IgG antibody response was higher in animals receiving macrophage inoculum against macrophage-derived Ehrlichia antigens, while it was significantly lower in the same animals against tick cell-derived Ehrlichia antigens. Deer infected with tick cell inoculum and tick transmission caused a higher antibody response to tick cell cultured bacterial antigens compared to the antibody response for macrophage cultured antigens for the same animals. The data demonstrate that the host cell-specific E. chaffeensis protein expression influences rickettsemia in a host and its acquisition by ticks. The data also reveal that tick cell-derived inoculum is similar to tick transmission with reduced rickettsemia, IgG response and tick acquisition of E. chaffeensis

Management Strategy for Congenital Choledochal Cyst with Co-existing Intrahepatic Dilation and Aberrant Bile Duct As Well As Other Complicated Biliary Anomalies

The purpose of this study was to investigate and discuss imaging methods and management strategies for congenital choledochal cyst with co-existing intrahepatic dilation and aberrant bile duct as well as other complicated biliary anomalies. In this study we reviewed and analyzed 72 patients with congenital choledochal cyst, ranging in age from 15 days to 12 years old and who were seen at our hospital during the past 12 years, from January 1993 to October 2005. The image manifestation and clinical significance of patients with coxisting intrahepatic biliary dilation and aberrant bile duct were carefully examined during operation via MRCP, cholangiography and choledochoscope. Twenty-two cases (30.1%) presented with intrahepatic bile duct dilation and 12 of these were of the cystic type. That is, the orifice of the dilated intrahepatic tract that converged into the common hepatic duct showed membrane or septum-like stenosis. In 10 cases the dilation tapered off from the porta hepatis to the initiating terminals of the intra-hepatic bile ducts and was not accompanied by stenosis. An aberrant bile duct was observed in 2 of the cases. In 3 cases, the right and left hepatic ducts converged at the choledochal cyst. In conclusion, the imaging methods for intrahepatic bile duct dilation possess important clinical significance. Further, for hepatojejunostomy with radical excision of a choledochal cyst, additional operative procedures for intrahepatic stenosis, possible bile duct malformation and pancreaticobiliary common duct calculi can potentially reduce postoperative complications

High-Throughput Screen Reveals sRNAs Regulating crRNA Biogenesis by Targeting CRISPR Leader to Repress Rho Termination

Discovery of CRISPR-Cas systems is one of paramount importance in the field of microbiology. Currently, how CRISPR-Cas systems are finely regulated remains to be defined. Here we use small regulatory RNA (sRNA) library to screen sRNAs targeting type I-F CRISPR-Cas system through proximity ligation by T4 RNA ligase and find 34 sRNAs linking to CRISPR loci. Among 34 sRNAs for potential regulators of CRISPR, sRNA pant463 and PhrS enhance CRISPR loci transcription, while pant391 represses their transcription. We identify PhrS as a regulator of CRISPR-Cas by binding CRISPR leaders to suppress Rho-dependent transcription termination. PhrS-mediated anti-termination facilitates CRISPR locus transcription to generate CRISPR RNA (crRNA) and subsequently promotes CRISPR-Cas adaptive immunity against bacteriophage invasion. Furthermore, this also exists in type I-C/-E CRISPR-Cas, suggesting general regulatory mechanisms in bacteria kingdom. Our findings identify sRNAs as important regulators of CRISPR-Cas, extending roles of sRNAs in controlling bacterial physiology by promoting CRISPR-Cas adaptation priming

Transmission Electron Microscopy Reveals Distinct Macrophage- and Tick Cell-Specific Morphological Stages of Ehrlichia chaffeensis

Background: Ehrlichia chaffeensis is an emerging tick-borne rickettsial pathogen responsible for human monocytic ehrlichiosis. Despite the induction of an active host immune response, the pathogen has evolved to persist in its vertebrate and tick hosts. Understanding how the organism progresses in tick and vertebrate host cells is critical in identifying effective strategies to block the pathogen transmission. Our recent molecular and proteomic studies revealed differences in numerous expressed proteins of the organism during its growth in different host environments. Methodology/Principal Findings: Transmission electron microscopy analysis was performed to assess morphological changes in the bacterium within macrophages and tick cells. The stages of pathogen progression observed included the attachment of the organism to the host cells, its engulfment and replication within a morulae by binary fission and release of the organisms from infected host cells by complete host cell lysis or by exocytosis. E. chaffeensis grown in tick cells was highly pleomorphic and appears to replicate by both binary fission and filamentous type cell divisions. The presence of Ehrlichia-like inclusions was also observed within the nucleus of both macrophages and tick cells. This observation was confirmed by confocal microscopy and immunoblot analysis. Conclusions/Significance: Morphological differences in the pathogen’s progression, replication, and processing within macrophages and tick cells provide further evidence that E. chaffeensis employs unique host-cell specific strategies in support of adaptation to vertebrate and tick cell environments

State of the climate in 2018

In 2018, the dominant greenhouse gases released into Earth’s atmosphere—carbon dioxide, methane, and nitrous oxide—continued their increase. The annual global average carbon dioxide concentration at Earth’s surface was 407.4 ± 0.1 ppm, the highest in the modern instrumental record and in ice core records dating back 800 000 years. Combined, greenhouse gases and several halogenated gases contribute just over 3 W m−2 to radiative forcing and represent a nearly 43% increase since 1990. Carbon dioxide is responsible for about 65% of this radiative forcing. With a weak La Niña in early 2018 transitioning to a weak El Niño by the year’s end, the global surface (land and ocean) temperature was the fourth highest on record, with only 2015 through 2017 being warmer. Several European countries reported record high annual temperatures. There were also more high, and fewer low, temperature extremes than in nearly all of the 68-year extremes record. Madagascar recorded a record daily temperature of 40.5°C in Morondava in March, while South Korea set its record high of 41.0°C in August in Hongcheon. Nawabshah, Pakistan, recorded its highest temperature of 50.2°C, which may be a new daily world record for April. Globally, the annual lower troposphere temperature was third to seventh highest, depending on the dataset analyzed. The lower stratospheric temperature was approximately fifth lowest. The 2018 Arctic land surface temperature was 1.2°C above the 1981–2010 average, tying for third highest in the 118-year record, following 2016 and 2017. June’s Arctic snow cover extent was almost half of what it was 35 years ago. Across Greenland, however, regional summer temperatures were generally below or near average. Additionally, a satellite survey of 47 glaciers in Greenland indicated a net increase in area for the first time since records began in 1999. Increasing permafrost temperatures were reported at most observation sites in the Arctic, with the overall increase of 0.1°–0.2°C between 2017 and 2018 being comparable to the highest rate of warming ever observed in the region. On 17 March, Arctic sea ice extent marked the second smallest annual maximum in the 38-year record, larger than only 2017. The minimum extent in 2018 was reached on 19 September and again on 23 September, tying 2008 and 2010 for the sixth lowest extent on record. The 23 September date tied 1997 as the latest sea ice minimum date on record. First-year ice now dominates the ice cover, comprising 77% of the March 2018 ice pack compared to 55% during the 1980s. Because thinner, younger ice is more vulnerable to melting out in summer, this shift in sea ice age has contributed to the decreasing trend in minimum ice extent. Regionally, Bering Sea ice extent was at record lows for almost the entire 2017/18 ice season. For the Antarctic continent as a whole, 2018 was warmer than average. On the highest points of the Antarctic Plateau, the automatic weather station Relay (74°S) broke or tied six monthly temperature records throughout the year, with August breaking its record by nearly 8°C. However, cool conditions in the western Bellingshausen Sea and Amundsen Sea sector contributed to a low melt season overall for 2017/18. High SSTs contributed to low summer sea ice extent in the Ross and Weddell Seas in 2018, underpinning the second lowest Antarctic summer minimum sea ice extent on record. Despite conducive conditions for its formation, the ozone hole at its maximum extent in September was near the 2000–18 mean, likely due to an ongoing slow decline in stratospheric chlorine monoxide concentration. Across the oceans, globally averaged SST decreased slightly since the record El Niño year of 2016 but was still far above the climatological mean. On average, SST is increasing at a rate of 0.10° ± 0.01°C decade−1 since 1950. The warming appeared largest in the tropical Indian Ocean and smallest in the North Pacific. The deeper ocean continues to warm year after year. For the seventh consecutive year, global annual mean sea level became the highest in the 26-year record, rising to 81 mm above the 1993 average. As anticipated in a warming climate, the hydrological cycle over the ocean is accelerating: dry regions are becoming drier and wet regions rainier. Closer to the equator, 95 named tropical storms were observed during 2018, well above the 1981–2010 average of 82. Eleven tropical cyclones reached Saffir–Simpson scale Category 5 intensity. North Atlantic Major Hurricane Michael’s landfall intensity of 140 kt was the fourth strongest for any continental U.S. hurricane landfall in the 168-year record. Michael caused more than 30 fatalities and $25 billion (U.S. dollars) in damages. In the western North Pacific, Super Typhoon Mangkhut led to 160 fatalities and$6 billion (U.S. dollars) in damages across the Philippines, Hong Kong, Macau, mainland China, Guam, and the Northern Mariana Islands. Tropical Storm Son-Tinh was responsible for 170 fatalities in Vietnam and Laos. Nearly all the islands of Micronesia experienced at least moderate impacts from various tropical cyclones. Across land, many areas around the globe received copious precipitation, notable at different time scales. Rodrigues and Réunion Island near southern Africa each reported their third wettest year on record. In Hawaii, 1262 mm precipitation at Waipā Gardens (Kauai) on 14–15 April set a new U.S. record for 24-h precipitation. In Brazil, the city of Belo Horizonte received nearly 75 mm of rain in just 20 minutes, nearly half its monthly average. Globally, fire activity during 2018 was the lowest since the start of the record in 1997, with a combined burned area of about 500 million hectares. This reinforced the long-term downward trend in fire emissions driven by changes in land use in frequently burning savannas. However, wildfires burned 3.5 million hectares across the United States, well above the 2000–10 average of 2.7 million hectares. Combined, U.S. wildfire damages for the 2017 and 2018 wildfire seasons exceeded $40 billion (U.S. dollars)

Aggregate-reactivation activity of the molecular chaperone ClpB from Ehrlichia chaffeensis

Citation: Zhang T, Kedzierska-Mieszkowska S, Liu H, Cheng C, Ganta RR, Zolkiewski M (2013) Aggregate-Reactivation Activity of the Molecular Chaperone ClpB from Ehrlichia chaffeensis. PLoS ONE 8(5): e62454. https://doi.org/10.1371/journal.pone.0062454Rickettsiale diseases, including human monocytic ehrlichiosis caused by Ehrlichia chaffeensis, are the second leading cause of the tick-borne infections in the USA and a growing health concern. Little is known about how E. chaffeensis survives the host-induced stress in vertebrate and tick hosts. A molecular chaperone ClpB from several microorganisms has been reported to reactivate aggregated proteins in cooperation with the co-chaperones DnaK/DnaJ/GrpE (KJE). In this study, we performed the first biochemical characterization of ClpB from E. chaffeensis. The transcript of E. chaffeensis ClpB (EhClpB) is strongly upregulated after infection of cultured macrophages and its level remains high during the Ehrlichia replicative stage. EhClpB forms ATP-dependent oligomers and catalyzes the ATP hydrolysis, similar to E. coli ClpB (EcClpB), but its ATPase activity is insensitive to the EcClpB activators, casein and poly-lysine. EhClpB in the presence of E. coli KJE efficiently reactivates the aggregated glucose-6-phosphate dehydrogenase (G6PDH) and firefly luciferase. Unlike EcClpB, which requires the co-chaperones for aggregate reactivation, EhClpB reactivates G6PDH even in the absence of KJE. Moreover, EhClpB is functionally distinct from EcClpB as evidenced by its failure to rescue a temperature-sensitive phenotype of the clpB-null E. coli. The clpB expression pattern during the E. chaffeensis infection progression correlates with the pathogen’s replicating stage inside host cells and suggests an essential role of the disaggregase activity of ClpB in the pathogen’s response to the host-induced stress. This study sets the stage for assessing the importance of the chaperone activity of ClpB for E. chaffeensis growth within the mammalian and tick hosts

Molecular Heterogeneity of Ehrlichia chaffeensis Isolates Determined by Sequence Analysis of the 28-Kilodalton Outer Membrane Protein Genes and Other Regions of the Genome

Ehrlichia chaffeensis, a tick-transmitted rickettsial agent, is responsible for human monocytic ehrlichiosis (HME). In this study, we genetically mapped 10 isolates obtained from HME patients. Sequence analysis of the 28-kDa outer membrane protein (OMP) multigene locus spanning 6 of the 22 tandemly arranged genes identified three distinct genetic groups with shared homology among isolates within each group. Isolates in Groups I and III contained six genes each, while Group II isolates had a gene deletion. There were two regions on the locus where novel gene deletion or insertion mutations occurred, resulting in the net loss of one gene in Group II isolates. Numerous nucleotide differences among genes in isolates of each group also were detected. The shared homology among isolates in each group for the 28-kDa OMP locus suggests the derivation of clonal lineages. Transcription and translation analysis of the locus revealed differences in the expressed genes of different group isolates. Analysis of the 120-kDa OMP gene and variable-length PCR target gene showed size variations resulting from loss or gain of long, direct repeats within the protein coding sequences. To our knowledge this is the first study that looked at several regions of the genome simultaneously, and we provide the first evidence of heterogeneity resulting from gene deletion and insertion mutations in the E. chaffeensis genome. Diversity in different genomic regions could be the result of a selection process or of independently evolved genes