Evolution, purification, and characterization of RC0497: a peptidoglycan amidase from the prototypical spotted fever species <i>Rickettsia conorii</i>

Patel, Jignesh; Narra, Hema P.; Sepuru, Krishna Mohan; Sahni, Abha; Golla, Sandhya R.; Sahni, Aishwarya; Singh, Amber; Schroeder, Casey L. C.; Chowdhury, Imran H.; Popov, Vsevolod L.; Sahni, Sanjeev K.

doi:10.1515/hsz-2018-0389

Cited by 6 publications

(7 citation statements)

References 66 publications

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“…Interestingly, RC0497 was determined to be the most abundantly expressed gene in both cell types (Tables 1 and 2 ). Recently, we have characterized RC0497 as an ampD domain containing N-acteylmuramoyl-L-alanine amidase involved in peptidoglycan hydrolysis and demonstrated its localization on the septal regions in dividing bacteria and on the membranes of vesicles protruding from the rickettsial cell wall [ 28 ]. A simultaneous study has further revealed that RC0497 is also secreted into the culture supernatants during infection of endothelial cells in vitro and is readily detectable in the serum of infected patients, projecting it as a promising candidate for the design and development of rapid diagnostics [ 29 ].…”

Section: Discussionmentioning

confidence: 99%

Comparative transcriptomic analysis of Rickettsia conorii during in vitro infection of human and tick host cells

et al. 2020

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Background Pathogenic Rickettsia species belonging to the spotted fever group are arthropod-borne, obligate intracellular bacteria which exhibit preferential tropism for host microvascular endothelium in the mammalian hosts, resulting in disease manifestations attributed primarily to endothelial damage or dysfunction. Although rickettsiae are known to undergo evolution through genomic reduction, the mechanisms by which these pathogens regulate their transcriptome to ensure survival in tick vectors and maintenance by transovarial/transstadial transmission, in contrast to their ability to cause debilitating infections in human hosts remain unknown. In this study, we compare the expression profiles of rickettsial sRNAome/transcriptome and determine the transcriptional start sites (TSSs) of R. conorii transcripts during in vitro infection of human and tick host cells. Results We performed deep sequencing on total RNA from Amblyomma americanum AAE2 cells and human microvascular endothelial cells (HMECs) infected with R. conorii. Strand-specific RNA sequencing of R. conorii transcripts revealed the expression 32 small RNAs (Rc_sR’s), which were preferentially expressed above the limit of detection during tick cell infection, and confirmed the expression of Rc_sR61, sR71, and sR74 by quantitative RT-PCR. Intriguingly, a total of 305 and 132 R. conorii coding genes were differentially upregulated (> 2-fold) in AAE2 cells and HMECs, respectively. Further, enrichment for primary transcripts by treatment with Terminator 5′-Phosphate-dependent Exonuclease resulted in the identification of 3903 and 2555 transcription start sites (TSSs), including 214 and 181 primary TSSs in R. conorii during the infection to tick and human host cells, respectively. Seventy-five coding genes exhibited different TSSs depending on the host environment. Finally, we also observed differential expression of 6S RNA during host-pathogen and vector-pathogen interactions in vitro, implicating an important role for this noncoding RNA in the regulation of rickettsial transcriptome depending on the supportive host niche. Conclusions In sum, the findings of this study authenticate the presence of novel Rc_sR’s in R. conorii, reveal the first evidence for differential expression of coding transcripts and utilization of alternate transcriptional start sites depending on the host niche, and implicate a role for 6S RNA in the regulation of coding transcriptome during tripartite host-pathogen-vector interactions.

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Section: Discussionmentioning

confidence: 99%

Comparative transcriptomic analysis of Rickettsia conorii during in vitro infection of human and tick host cells

et al. 2020

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“…Infectivity titers of purified stocks were estimated by citrate synthase ( gltA )-based quantitative PCR and/or plaque-formation assay [ 30 , 31 ]. HMECs were infected with R. rickettsii at an MOI (multiplicity of infection) of approximately 20 intracellular rickettsiae per cell following our standard laboratory protocols [ 30 , 32 ]. The viability of both mock-infected (controls) and R. rickettsii -infected HMECs were monitored microscopically.…”

Section: Methodsmentioning

confidence: 99%

Sensing the Messenger: Potential Roles of Cyclic-di-GMP in Rickettsial Pathogenesis

Narra

Sahni

Sepuru

et al. 2022

IJMS

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Pathogenic bacteria causing human rickettsioses, transmitted in nature by arthropod vectors, primarily infect vascular endothelial cells lining the blood vessels, resulting in ‘endothelial activation’ and onset of innate immune responses. Nucleotide second messengers are long presumed to be the stimulators of type I interferons, of which bacterial cyclic-di-GMP (c-di-GMP) has been implicated in multiple signaling pathways governing communication with other bacteria and host cells, yet its importance in the context of rickettsial interactions with the host has not been investigated. Here, we report that all rickettsial genomes encode a putative diguanylate cyclase pleD, responsible for the synthesis of c-di-GMP. In silico analysis suggests that although the domain architecture of PleD is apparently well-conserved among different rickettsiae, the protein composition and sequences likely vary. Interestingly, cloning and sequencing of the pleD gene from virulent (Sheila Smith) and avirulent (Iowa) strains of R. rickettsii reveals a nonsynonymous substitution, resulting in an amino acid change (methionine to isoleucine) at position 236. Additionally, a previously reported 5-bp insertion in the genomic sequence coding for pleD (NCBI accession: NC_009882) was not present in the sequence of our cloned pleD from R. rickettsii strain Sheila Smith. In vitro infection of HMECs with R. rickettsii (Sheila Smith), but not R. rickettsii (Iowa), resulted in dynamic changes in the levels of pleD up to 24 h post-infection. These findings thus provide the first evidence for the potentially important role(s) of c-di-GMP in the determination of host-cell responses to pathogenic rickettsiae. Further studies into molecular mechanisms through which rickettsial c-di-GMP might regulate pathogen virulence and host responses should uncover the contributions of this versatile bacterial second messenger in disease pathogenesis and immunity to human rickettsioses.

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“…Nowadays, research on transcriptomics of R. conorii aims to shed light on the potential regulatory role of noncoding RNA during the course of rickettsial infections [66,72]. Furthermore, proteomic studies have identified a new protein molecule implicated in various host-rickettsial interactions, that could have a potential diagnostic, as well as prognostic, value [73,74]. Nevertheless, many aspects of R. conorii infection pathogenesis remain obscure.…”

Section: Pathogenesismentioning

confidence: 99%

Mediterranean Spotted Fever: Current Knowledge and Recent Advances

Spernovasilis

Markaki²,

Papadakis

et al. 2021

TropicalMed

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Mediterranean spotted fever (MSF) is an emerging tick-borne rickettsiosis of the spotted fever group (SFG), endemic in the Mediterranean basin. By virtue of technological innovations in molecular genetics, it has been determined that the causative agent of MSF is Rickettsia conorii subspecies conorii. The arthropod vector of this bacterium is the brown dog tick Rhipicephalus sanguineus. The true nature of the reservoir of R. conorii conorii has not been completely deciphered yet, although many authors theorize that the canine population, other mammals, and the ticks themselves could potentially contribute as reservoirs. Typical symptoms of MSF include fever, maculopapular rash, and a characteristic eschar (“tache noire”). Atypical clinical features and severe multi-organ complications may also be present. All of these manifestations arise from the disseminated infection of the endothelium by R. conorii conorii. Several methods exist for the diagnosis of MSF. Serological tests are widely used and molecular techniques have become increasingly available. Doxycycline remains the treatment of choice, while preventive measures are focused on modification of human behavior and vector control strategies. The purpose of this review is to summarize the current knowledge on the epidemiology, pathogenesis, clinical features, diagnosis, and treatment of MSF.

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Evolution, purification, and characterization of RC0497: a peptidoglycan amidase from the prototypical spotted fever species Rickettsia conorii

Cited by 6 publications

References 66 publications

Comparative transcriptomic analysis of Rickettsia conorii during in vitro infection of human and tick host cells

Comparative transcriptomic analysis of Rickettsia conorii during in vitro infection of human and tick host cells

Sensing the Messenger: Potential Roles of Cyclic-di-GMP in Rickettsial Pathogenesis

Mediterranean Spotted Fever: Current Knowledge and Recent Advances

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