Research

12 pages, 1001 KiB

Open AccessArticle

Identification, Baculoviral Expression, and Biochemical Characterization of a Novel Cholinesterase of Amblyomma americanum (Acari: Ixodidae)

by Kevin B. Temeyer, Kristie G. Schlechte, Aaron D. Gross and Kimberly H. Lohmeyer

Int. J. Mol. Sci. 2023, 24(9), 7681; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24097681 - 22 Apr 2023

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Abstract

A cDNA encoding a novel cholinesterase (ChE, EC 3.1.1.8) from the larvae of Amblyomma americanum (Linnaeus) was identified, sequenced, and expressed in Sf21 insect cell culture using the baculoviral expression vector pBlueBac4.5/V5-His. The open reading frame (1746 nucleotides) of the cDNA encoded 581 [...] Read more.

A cDNA encoding a novel cholinesterase (ChE, EC 3.1.1.8) from the larvae of Amblyomma americanum (Linnaeus) was identified, sequenced, and expressed in Sf21 insect cell culture using the baculoviral expression vector pBlueBac4.5/V5-His. The open reading frame (1746 nucleotides) of the cDNA encoded 581 amino acids beginning with the initiation codon. Identical cDNA sequences were amplified from the total RNA of adult tick synganglion and salivary gland, strongly suggesting expression in both tick synganglion and saliva. The recombinant enzyme (rAaChE1) was highly sensitive to eserine and BW284c51, relatively insensitive to tetraisopropyl pyrophosphoramide (iso-OMPA) and ethopropazine, and hydrolyzed butyrylthiocholine (BuTCh) 5.7 times as fast as acetylthiocholine (ATCh) at 120 µM, with calculated K_M values for acetylthiocholine (ATCh) and butyrylthiocholine of 6.39 µM and 14.18 µM, respectively. The recombinant enzyme was highly sensitive to inhibition by malaoxon, paraoxon, and coroxon in either substrate. Western blots using polyclonal rabbit antibody produced by immunization with a peptide specific for rAaChE1 exhibited reactivity in salivary and synganglial extract blots, indicating the presence of AaChE1 antigenic protein. Total cholinesterase activities of synganglial or salivary gland extracts from adult ticks exhibited biochemical properties very different from the expressed rAaACh1 enzyme, evidencing the substantial presence of additional cholinesterase activities in tick synganglion and saliva. The biological function of AaChE1 remains to be elucidated, but its presence in tick saliva is suggestive of functions in hydrolysis of cholinergic substrates present in the large blood mean and potential involvement in the modulation of host immune responses to tick feeding and introduced pathogens. Full article

(This article belongs to the Special Issue Human Animal Tick-Borne Diseases)

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14 pages, 2636 KiB

Open AccessArticle

A Small Non-Coding RNA Mediates Transcript Stability and Expression of Cytochrome bd Ubiquinol Oxidase Subunit I in Rickettsia conorii

by Hema P. Narra, Jessica Alsing, Abha Sahni, Michelle Montini, Yasim Zafar and Sanjeev K. Sahni

Int. J. Mol. Sci. 2023, 24(4), 4008; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24044008 - 16 Feb 2023

Cited by 1 | Viewed by 1248

Abstract

Small regulatory RNAs (sRNAs) are now widely recognized for their role in the post-transcriptional regulation of bacterial virulence and growth. We have previously demonstrated the biogenesis and differential expression of several sRNAs in Rickettsia conorii during interactions with the human host and arthropod [...] Read more.

Small regulatory RNAs (sRNAs) are now widely recognized for their role in the post-transcriptional regulation of bacterial virulence and growth. We have previously demonstrated the biogenesis and differential expression of several sRNAs in Rickettsia conorii during interactions with the human host and arthropod vector, as well as the in vitro binding of Rickettsia conorii sRNA Rc_sR42 to bicistronic cytochrome bd ubiquinol oxidase subunits I and II (cydAB) mRNA. However, the mechanism of regulation and the effect of sRNA binding on the stability of the cydAB bicistronic transcript and the expression of the cydA and cydB genes are still unknown. In this study, we determined the expression dynamics of Rc_sR42 and its cognate target genes, cydA and cydB, in mouse lung and brain tissues during R. conorii infection in vivo and employed fluorescent and reporter assays to decode the role of sRNA in regulating cognate gene transcripts. Quantitative RT-PCR revealed significant changes in the expression of sRNA and its cognate target gene transcripts during R. conorii infection in vivo, and a greater abundance of these transcripts was observed in the lungs compared to brain tissue. Interestingly, while Rc_sR42 and cydA exhibited similar patterns of change in their expression, indicating the influence of sRNA on the mRNA target, the expression of cydB was independent of sRNA expression. Further, we constructed reporter plasmids of sRNA and cydAB bicistronic mRNA to decipher the role of sRNA on CydA and CydB expression. We observed increased expression of CydA in the presence of sRNA but detected no change in CydB expression in the presence or absence of sRNA. In sum, our results demonstrate that the binding of Rc_sR42 is required for the regulation of cydA but not cydB. Further studies on understanding the influence of this interaction on the mammalian host and tick vector during R. conorii infection are in progress. Full article

(This article belongs to the Special Issue Human Animal Tick-Borne Diseases)

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12 pages, 1702 KiB

Open AccessCommunication

Detection and Genotypic Analysis of Anaplasma bovis and A. phagocytophilum in Horse Blood and Lung Tissue

by Min-Goo Seo, In-Ohk Ouh and Dongmi Kwak

Int. J. Mol. Sci. 2023, 24(4), 3239; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24043239 - 07 Feb 2023

Cited by 2 | Viewed by 1398

Abstract

A clinical case of Anaplasma bovis was reported for the first time in our previous study (2019) in a horse, a nondefinitive host. Although A. bovis is a ruminant and not a zoonotic pathogen, it is responsible for persistent infections in horses. In [...] Read more.

A clinical case of Anaplasma bovis was reported for the first time in our previous study (2019) in a horse, a nondefinitive host. Although A. bovis is a ruminant and not a zoonotic pathogen, it is responsible for persistent infections in horses. In this follow-up study, the prevalence of Anaplasma spp., including A. bovis, was assessed in horse blood and lung tissue samples to fully understand Anaplasma spp. pathogen distribution and the potential risk factors of infection. Among 1696 samples, including 1433 blood samples from farms nationwide and 263 lung tissue samples from horse abattoirs on Jeju Island, a total of 29 samples (1.7%) tested positive for A. bovis and 31 (1.8%) samples tested positive for A. phagocytophilum, as determined by 16S rRNA nucleotide sequencing and restriction fragment length polymorphism. This study is the first to detect A. bovis infection in horse lung tissue samples. Further studies are needed to clarify the comparison of sample types within cohorts. Although the clinical significance of Anaplasma infection was not evaluated in this study, our results emphasize the need to clarify the host tropism and genetic divergence of Anaplasma to enable the development of effective prevention and control measures through broad epidemiological studies. Full article

(This article belongs to the Special Issue Human Animal Tick-Borne Diseases)

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11 pages, 9757 KiB

Open AccessArticle

Emergence of Anaplasma Species Related to A. phagocytophilum and A. platys in Senegal

by Rosanna Zobba, Claudio Murgia, Mustapha Dahmani, Oleg Mediannikov, Bernard Davoust, Roberta Piredda, Eleonora Schianchi, Alessandra Scagliarini, Marco Pittau and Alberto Alberti

Int. J. Mol. Sci. 2023, 24(1), 35; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24010035 - 20 Dec 2022

Cited by 4 | Viewed by 1235

Abstract

The genus Anaplasma (Anaplasmataceae, Rickettsiales) includes tick-transmitted bacterial species of importance to both veterinary and human medicine. Apart from the traditionally recognized six Anaplasma species (A. phagocytophilum, A. platys, A. bovis, A. ovis, A. centrale, A. [...] Read more.

The genus Anaplasma (Anaplasmataceae, Rickettsiales) includes tick-transmitted bacterial species of importance to both veterinary and human medicine. Apart from the traditionally recognized six Anaplasma species (A. phagocytophilum, A. platys, A. bovis, A. ovis, A. centrale, A. marginale), novel strains and candidate species, also of relevance to veterinary and human medicine, are emerging worldwide. Although species related to the zoonotic A. platys and A. phagocytophilum have been reported in several African and European Mediterranean countries, data on the presence of these species in sub-Saharan countries are still lacking. This manuscript reports the investigation of Anaplasma strains related to zoonotic species in ruminants in Senegal by combining different molecular tests and phylogenetic approaches. The results demonstrated a recent introduction of Candidatus (Ca) Anaplasma turritanum, a species related to the pathogenic A. platys, possibly originating by founder effect. Further, novel undetected strains related to Candidatus (Ca) Anaplasma cinensis were detected in cattle. Based on groEL and gltA molecular comparisons, we propose including these latter strains into the Candidatus (Ca) Anaplasma africanum species. Finally, we also report the emergence of Candidatus (Ca) A. boleense in Senegal. Collectively, results confirm that Anaplasma species diversity is greater than expected and should be further investigated, and that Anaplasma routine diagnostic procedures and epidemiological surveillance should take into account specificity issues raised by the presence of these novel strains, suggesting the use of a One Health approach for the management of Anaplasmataceae in sub-Saharan Africa. Full article

(This article belongs to the Special Issue Human Animal Tick-Borne Diseases)

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14 pages, 2685 KiB

Open AccessArticle

Analysis of the Type 4 Effectome across the Genus Rickettsia

by Joseph A. Aspinwall and Kelly A. Brayton

Int. J. Mol. Sci. 2022, 23(24), 15513; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232415513 - 08 Dec 2022

Cited by 1 | Viewed by 1355

Abstract

Rickettsia are obligate intracellular bacteria primarily carried by arthropod hosts. The genus Rickettsia contains several vertebrate pathogens vectored by hematophagous arthropods. Despite the potential for disease, our understanding of Rickettsias are limited by the difficulties associated with growing and manipulating obligate intracellular bacteria. [...] Read more.

Rickettsia are obligate intracellular bacteria primarily carried by arthropod hosts. The genus Rickettsia contains several vertebrate pathogens vectored by hematophagous arthropods. Despite the potential for disease, our understanding of Rickettsias are limited by the difficulties associated with growing and manipulating obligate intracellular bacteria. To aid with this, our lab conducted an analysis of eight genomes and three plasmids from across the genus Rickettsia. Using OPT4e, a learning algorithm-based program designed to identify effector proteins secreted by the type 4 secretion system, we generated a putative effectome for the genus. We then consolidated effectors into homolog sets to identify effectors unique to Rickettsia with different life strategies or evolutionary histories. We also compared predicted effectors to non-effectors for differences in G+C content and gene splitting. Based on this analysis, we predicted 1571 effectors across the genus, resulting in 604 homolog sets. Each species had unique homolog sets, while 42 were present in all eight species analyzed. Effectors were flagged in association with pathogenic, tick and flea-borne Rickettsia. Predicted effectors also varied in G+C content and frequency of gene splitting as compared to non-effectors. Species effector repertoires show signs of expansion, degradation, and horizontal acquisition associated with lifestyle and lineage. Full article

(This article belongs to the Special Issue Human Animal Tick-Borne Diseases)

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19 pages, 4265 KiB

Open AccessArticle

Evaluating EcxR for Its Possible Role in Ehrlichia chaffeensis Gene Regulation

by Huitao Liu, Cheyenne A. Knox, Laxmi U. M. R. Jakkula, Ying Wang, Lalitha Peddireddi and Roman R. Ganta

Int. J. Mol. Sci. 2022, 23(21), 12719; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232112719 - 22 Oct 2022

Cited by 1 | Viewed by 1114

Abstract

Ehrlichia chaffeensis, a tick-transmitted intraphagosomal bacterium, is the causative agent of human monocytic ehrlichiosis. The pathogen also infects several other vertebrate hosts. E. chaffeensis has a biphasic developmental cycle during its growth in vertebrate monocytes/macrophages and invertebrate tick cells. Host- and vector-specific [...] Read more.

Ehrlichia chaffeensis, a tick-transmitted intraphagosomal bacterium, is the causative agent of human monocytic ehrlichiosis. The pathogen also infects several other vertebrate hosts. E. chaffeensis has a biphasic developmental cycle during its growth in vertebrate monocytes/macrophages and invertebrate tick cells. Host- and vector-specific differences in the gene expression from many genes of E. chaffeensis are well documented. It is unclear how the organism regulates gene expression during its developmental cycle and for its adaptation to vertebrate and tick host cell environments. We previously mapped promoters of several E. chaffeensis genes which are recognized by its only two sigma factors: σ³² and σ⁷⁰. In the current study, we investigated in assessing five predicted E. chaffeensis transcription regulators; EcxR, CtrA, MerR, HU and Tr1 for their possible roles in regulating the pathogen gene expression. Promoter segments of three genes each transcribed with the RNA polymerase containing σ⁷⁰ (HU, P28-Omp14 and P28-Omp19) and σ³² (ClpB, DnaK and GroES/L) were evaluated by employing multiple independent molecular methods. We report that EcxR binds to all six promoters tested. Promoter-specific binding of EcxR to several gene promoters results in varying levels of gene expression enhancement. This is the first detailed molecular characterization of transcription regulators where we identified EcxR as a gene regulator having multiple promoter-specific interactions. Full article

(This article belongs to the Special Issue Human Animal Tick-Borne Diseases)

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19 pages, 30206 KiB

Open AccessArticle

Iron Reduction in Dermacentor andersoni Tick Cells Inhibits Anaplasma marginale Replication

by Muna Salem M. Solyman, Jessica Ujczo, Kelly A. Brayton, Dana K. Shaw, David A. Schneider and Susan M. Noh

Int. J. Mol. Sci. 2022, 23(7), 3941; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23073941 - 01 Apr 2022

Cited by 1 | Viewed by 1946

Abstract

Anaplasma spp. are obligate intracellular, tick-borne, bacterial pathogens that cause bovine and human anaplasmosis. We lack tools to prevent these diseases in part due to major knowledge gaps in our fundamental understanding of the tick–pathogen interface, including the requirement for and molecules involved [...] Read more.

Anaplasma spp. are obligate intracellular, tick-borne, bacterial pathogens that cause bovine and human anaplasmosis. We lack tools to prevent these diseases in part due to major knowledge gaps in our fundamental understanding of the tick–pathogen interface, including the requirement for and molecules involved in iron transport during tick colonization. We determine that iron is required for the pathogen Anaplasma marginale, which causes bovine anaplasmosis, to replicate in Dermacentor andersoni tick cells. Using bioinformatics and protein modeling, we identified three orthologs of the Gram-negative siderophore-independent iron uptake system, FbpABC. Am069, the A. marginale ortholog of FbpA, lacks predicted iron-binding residues according to the NCBI conserved domain database. However, according to protein modeling, the best structural orthologs of Am069 are iron transport proteins from Cyanobacteria and Campylobacterjejuni. We then determined that all three A. marginale genes are modestly differentially expressed in response to altered host cell iron levels, despite the lack of a Ferric uptake regulator or operon structure. This work is foundational for building a mechanistic understanding of iron uptake, which could lead to interventions to prevent bovine and human anaplasmosis. Full article

(This article belongs to the Special Issue Human Animal Tick-Borne Diseases)

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17 pages, 2304 KiB

Open AccessArticle

Rickettsial Pathogen Perturbs Tick Circadian Gene to Infect the Vertebrate Host

by Supreet Khanal, Vikas Taank, John F. Anderson, Hameeda Sultana and Girish Neelakanta

Int. J. Mol. Sci. 2022, 23(7), 3545; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23073545 - 24 Mar 2022

Cited by 8 | Viewed by 2368

Abstract

Ixodes scapularis is a medically important tick that transmits several microbes to humans, including rickettsial pathogen Anaplasma phagocytophilum. In nature, these ticks encounter several abiotic factors including changes in temperature, humidity, and light. Many organisms use endogenously generated circadian pathways to [...] Read more.

Ixodes scapularis is a medically important tick that transmits several microbes to humans, including rickettsial pathogen Anaplasma phagocytophilum. In nature, these ticks encounter several abiotic factors including changes in temperature, humidity, and light. Many organisms use endogenously generated circadian pathways to encounter abiotic factors. In this study, we provide evidence for the first time to show that A. phagocytophilum modulates the arthropod circadian gene for its transmission to the vertebrate host. We noted a circadian oscillation in the expression of arthropod clock, bmal1, period and timeless genes when ticks or tick cells were exposed to alternate 12 h light: 12 h dark conditions. Moreover, A. phagocytophilum significantly modulates the oscillation pattern of expression of these genes. In addition, increased levels of clock and bmal1 and decreased expression of Toll and JAK/STAT pathway immune genes such as pelle and jak, respectively, were noted during A. phagocytophilum transmission from ticks to the vertebrate host. RNAi-mediated knockdown of clock gene expression in ticks resulted in the reduced expression of jak and pelle that increased bacterial transmission from ticks to the murine host. Furthermore, clock-deficient ticks fed late and had less engorgement weights. These results indicate an important role for circadian modulation of tick gene expression that is critical for arthropod blood feeding and transmission of pathogens from vector to the vertebrate host. Full article

(This article belongs to the Special Issue Human Animal Tick-Borne Diseases)

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