Hema P. Narra

Small Regulatory RNAs of Rickettsia conorii

Small regulatory RNAs comprise critically important modulators of gene expression in bacteria, yet very little is known about their prevalence and functions in Rickettsia species. R. conorii, the causative agent of Mediterranean spotted fever, is a tick-borne pathogen that primarily infects microvascular endothelium in humans. We have determined the transcriptional landscape of R. conorii during infection of Human Microvascular Endothelial Cells (HMECs) by strand-specific RNA sequencing to identify 4 riboswitches, 13 trans-acting (intergenic), and 22 cis-acting (antisense) small RNAs (termed ‘Rc_sR’s). Independent expression of four novel trans-acting sRNAs (Rc_sR31, Rc_sR33, Rc_sR35, and Rc_sR42) and known bacterial sRNAs (6S, RNaseP_bact_a, ffs, and α-tmRNA) was next confirmed by Northern hybridization. Comparative analysis during infection of HMECs vis-à-vis tick AAE2 cells revealed significantly higher expression of Rc_sR35 and Rc_sR42 in HMECs, whereas Rc_sR31 and Rc_sR33 were expressed at similar levels in both cell types. We further predicted a total of 502 genes involved in all important biological processes as potential targets of Rc_sRs and validated the interaction of Rc_sR42 with cydA (cytochrome d ubiquinol oxidase subunit I). Our findings constitute the first evidence of the existence of post-transcriptional riboregulatory mechanisms in R. conorii and interactions between a novel Rc_sR and its target mRNA.

Identification and Characterization of Novel Small RNAs in Rickettsia prowazekii

Emerging evidence implicates a critically important role for bacterial small RNAs (sRNAs) as post-transcriptional regulators of physiology, metabolism, stress/adaptive responses, and virulence, but the roles of sRNAs in pathogenic Rickettsia species remain poorly understood. Here, we report on the identification of both novel and well-known bacterial sRNAs in Rickettsia prowazekii, known to cause epidemic typhus in humans. RNA sequencing of human microvascular endothelial cells (HMECs), the preferred targets during human rickettsioses, infected with R. prowazekii revealed the presence of 35 trans-acting and 23 cis-acting sRNAs, respectively. Of these, expression of two trans-acting (Rp_sR17 and Rp_sR60) and one cis-acting (Rp_sR47) novel sRNAs and four well-characterized bacterial sRNAs (RNaseP_bact_a, α-tmRNA, 4.5S RNA, 6S RNA) was further confirmed by Northern blot or RT-PCR analyses. The transcriptional start sites of five novel rickettsial sRNAs and 6S RNA were next determined using 5′ RLM-RACE yielding evidence for their independent biogenesis in R. prowazekii. Finally, computational approaches were employed to determine the secondary structures and potential mRNA targets of novel sRNAs. Together, these results establish the presence and expression of sRNAs in R. prowazekii during host cell infection and suggest potential functional roles for these important post-transcriptional regulators in rickettsial biology and pathogenesis.

Transcriptional profiling of Rickettsia prowazekii coding and non-coding transcripts during in vitro host-pathogen and vector-pathogen interactions

Natural pathogen transmission of Rickettsia prowazekii, the etiologic agent of epidemic typhus, to humans is associated with arthropods, including human body lice, ticks, and ectoparasites of eastern flying squirrel. Recently, we have documented the presence of small RNAs in Rickettsia species and expression of R. prowazekii sRNAs during infection of cultured human microvascular endothelial cells (HMECs), which represent the primary target cells during human infections. Bacterial noncoding transcripts are now well established as critical post-transcriptional regulators of virulence and adaptation mechanisms in varying host environments. Despite their importance, little is known about the expression profile and regulatory activities of R. prowazekii sRNAs (Rp_sRs) in different host cells encountered as part of the natural life-cycle. To investigate the sRNA expression profile of R. prowazekii during infection of arthropod host cells, we employed an approach combining in vitro infection, bioinformatics, RNA sequencing, and PCR-based quantitation. Global analysis of R. prowazekii transcriptome by strand-specific RNA sequencing enabled us to identify 67 cis-acting (antisense) and 26 trans-acting (intergenic) Rp_sRs expressed during the infection of Amblyomma americanum (AAE2) cells. Comparative evaluation of expression during R. prowazekii infection of HMECs and AAE2 cells by quantitative RT-PCR demonstrated significantly higher expression of four selected Rp_sRs in tick AAE2 cells. Examination of the coding transcriptome revealed differential up-regulation of >150 rickettsial genes in either HMECs or AAE2 cells and yielded evidence for host cell-dependent utilization of alternative transcription start sites by 18 rickettsial genes. Our results thus suggest noticeable differences in the expression of both Rp_sRs as well as the coding transcriptome and the exploitation of multiple transcription initiation sites for select genes during the infection of human endothelium and tick vector cells as the host and yield new insights into rickettsial virulence and transmission mechanisms.

MicroRNA Signature of Human Microvascular Endothelium Infected with Rickettsia rickettsii

MicroRNAs (miRNAs) mediate gene silencing by destabilization and/or translational repression of target mRNA. Infection of human microvascular endothelial cells as primary targets of Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever, triggers host responses appertaining to alterations in cellular gene expression. Microarray-based profiling of endothelial cells infected with R. rickettsii for 3 or 24 h revealed differential expression of 33 miRNAs, of which miRNAs129-5p, 200a-3p, 297, 200b-3p, and 595 were identified as the top five up-regulated miRNAs (5 to 20-fold, p ≤ 0.01) and miRNAs 301b-3p, 548a-3p, and 377-3p were down-regulated (2 to 3-fold, p ≤ 0.01). Changes in the expression of selected miRNAs were confirmed by q-RT-PCR in both in vitro and in vivo models of infection. As potential targets, expression of genes encoding NOTCH1, SMAD2, SMAD3, RIN2, SOD1, and SOD2 was either positively or negatively regulated. Using a miRNA-specific mimic or inhibitor, NOTCH1 was determined to be a target of miRNA 200a-3p in R. rickettsii-infected human dermal microvascular endothelial cells (HMECs). Predictive interactome mapping suggested the potential for miRNA-mediated modulation of regulatory gene networks underlying important host cell signaling pathways. This first demonstration of altered endothelial miRNA expression provides new insights into regulatory elements governing mechanisms of host responses and pathogenesis during human rickettsial infections.

Endothelial Activation and Injury: The Mechanisms of Rickettsial Vasculiti

Rickettsial infections continue to pose a significant health threat to humans of all ages across the globe. Rickettsiae are obligate intracellular, Gram-negative bacteria with tropism for microvascular endothelium in humans during natural transmission from arthropod vectors and in experimental mouse models of infection. Consequently, pathogenesis of rickettsioses primarily involves endothelial activation, inflammation, dysfunction, and compromised barrier integrity, collectively referred to as “rickettsial vasculitis.” A procoagulative phenotype may develop in rickettsial infection, resulting in the formation of hemostatic plugs at the sites of severe vascular damage, but only rare occurrence of disseminated intravascular coagulation. This chapter summarizes unique features of pathogenic Rickettsia species, the spectrum of rickettsial diseases, and recent advancements in cellular and molecular mechanisms of pathogenesis with primary focus on endothelial cell responses to infection. Comprehensive understanding of interactions between this unique group of cytoplasmic pathogens and the host vasculature should facilitate the development of new therapeutics to prevent or reverse endothelial dysfunction and vascular leakage associated with rickettsial infections.

Expression Profiling of Long Noncoding RNA Splice Variants in Human Microvascular Endothelial Cells: Lipopolysaccharide Effects In Vitro

Endothelial cell interactions with lipopolysaccharide (LPS) involve both activating and repressing signals resulting in pronounced alterations in their transcriptome and proteome. Noncoding RNAs are now appreciated as posttranscriptional and translational regulators of cellular signaling and responses, but their expression status and roles during endothelial interactions with LPS are not well understood. We report on the expression profile of long noncoding (lnc) RNAs of human microvascular endothelial cells in response to LPS. We have identified a total of 10,781 and 8310 lncRNA transcripts displaying either positive or negative regulation of expression, respectively, at 3 and 24 h posttreatment. A majority of LPS-induced lncRNAs are multiexonic and distributed across the genome as evidenced by their presence on all chromosomes. Present among these are a total of 44 lncRNAs with known regulatory functions, of which 41 multiexonic lncRNAs have multiple splice variants. We have further validated splice variant-specific expression of EGO (NONHSAT087634) and HOTAIRM1 (NONHSAT119666) at 3 h and significant upregulation of lnc-IL7R at 24 h. This study illustrates the genome-wide regulation of endothelial lncRNA splice variants in response to LPS and provides a foundation for further investigations of differentially expressed lncRNA transcripts in endothelial responses to LPS and pathophysiology of sepsis/septic shock.

CRISPR/Cas9-mediated gene deletion of the ompA gene in an Enterobacter gut symbiont impairs biofilm formation and reduces gut colonization of Aedes aegypti mosquitoes

Symbiotic bacteria are pervasive in mosquitoes and their presence can influence development, reproduction, and immunity of their host. It is evident that environmental and host genetic factors contribute in shaping the microbiome of mosquitoes, but we have a poor understanding regarding how bacterial genetics affects colonization of the mosquito gut. While CRISPR/Cas9 gene editing is a powerful tool to modify bacterial genomes this approach has yet to be applied to insect symbionts. To demonstrate that gene editing can be completed in non-model bacterial species isolated from insects and to investigate the role of bacterial genes in gut colonization, we mutated the outer membrane protein A (ompA) gene of an Enterobacter symbiont using the CRISPR/Cas9 system. The ΔompA mutant had an impaired ability to form biofilms and poorly infected Ae. aegypti when reared in a mono-association under gnotobiotic conditions. In adults, the mutant had a significantly reduced infection prevalence compared to the wild type or complement strains, while no differences in prevalence were seen in larvae, suggesting bacterial genetic factors are important for adult gut colonization. Integration of genes (antibiotic resistance and fluorescent markers) into the symbiont genome demonstrated this technology can be exploited to develop novel symbiotic control strategies to interfere with arboviral pathogens such Chikungunya, Zika and Yellow fever viruses transmitted by Aedes mosquitoes. Our results shed insights onto the role of ompA gene in host-microbe interactions in Ae. aegypti and confirm that CRISPR/Cas9 gene editing can be employed for genetic manipulation of non-model gut microbes. Importance CRISPR/Cas9 gene editing approaches have revolutionized several biological fields, however despite their applicability for altering bacterial genomes, few studies use this technology in microbes that associated with eukaryotic hosts. Here we use this editing approach to knockout a gene encoding a membrane protein in an Enterobacter isolated from Aedes mosquitoes and show this gene is essential for biofilm formation and promotes bacteria colonization of the gut. A reduced bacterial load of the mutant compared to the wild type or complement strains, was seen in both larval and adult mosquitoes, however this was most evident in adults, likely due differences in the mode of acquisition of microbes at each life stage. Our work extends CRISPR/Cas9 genetic manipulation into a new bacterial species, and in conjunction with other studies, suggests that members within Enterobacteriaceae are amenable to genome engineering by this approach. This study will facilitate the development of novel microbial-based approaches to mitigate mosquito-borne disease.

Fibroblast growth factor receptor-1 mediates internalization of pathogenic spotted fever rickettsiae into host endothelium

Rickettsial infections continue to cause serious morbidity and mortality in severe human cases around the world. Host cell adhesion and invasion is an essential requisite for intracellular growth, replication, and subsequent dissemination of pathogenic rickettsiae. Heparan sulfate proteoglycans [HSPGs] facilitate the interactions between fibroblast growth factor(s) and their tyrosine kinase receptors resulting in receptor dimerization/activation and have been implicated in bacterial adhesion to target host cells. In the present study, we have investigated the contributions of fibroblast growth factor receptors [FGFRs] in rickettsial entry into the host cells. Inhibition of HSPGs by heparinase and FGFRs by AZD4547 (a selective small-molecule inhibitor) results in significant reduction in rickettsial internalization into cultured human microvascular endothelial cells (ECs), which represent the primary targets of pathogenic rickettsiae during human infections. Administration of AZD4547 during R. conorii infection in a murine model of endothelial-target spotted fever rickettsiosis also diminishes pulmonary rickettsial burden in comparison to mock-treated controls. Silencing of FGFR1 expression using a small interfering RNA also leads to similar inhibition of R. rickettsii invasion into ECs. Consistent with these findings, R. rickettsii infection of ECs also results in phosphorylation of tyrosine 653/654, suggesting activation of FGFR1. Using isobaric tag for relative and absolute quantitation [iTRAQ]-based proteomics approach, we further demonstrate association of β-peptide of rickettsial outer membrane protein OmpA with FGFR1. Mechanistically, FGFR1 binds to caveolin-1 and mediates bacterial entry via caveolin-1 dependent endocytosis. Together, these results identify host cell FGFR1 and rickettsial OmpA as another novel receptor-ligand pair contributing to the internalization of pathogenic rickettsiae into host endothelial cells and the potential application of FGFR-inhibitor drugs as adjunct therapeutics against spotted fever rickettsioses.

Human Rickettsioses: Host Response and Molecular Pathogenesis

Rickettsia are medically relevant obligately intracellular bacterial pathogens that cause potentially severe disease. As the causative agents of epidemic typhus and Rocky Mountain spotted fever, these neglected pathogens have had significant impacts on society with mortality rates reaching upwards of 50 %. Antibiotic therapies are often delayed, as initial diagnosis is difficult and easily confused with viral infections. This chapter will address Rickettsia and host interactions such as adhesion to and invasion of host target cells, endothelial pathogenesis, and immune response to rickettsioses. Further, the chapter will provide an overview of rickettsial genetics, metabolism, and secretion systems and their relationship to molecular pathogenesis.