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Raghavan, R.

Publications and source records attributed to Raghavan, R..

18 recordsLinked to original sources
bioRxiv2026

Stepwise assembly of virulence-associated traits in the intracellular pathogen Coxiella burnetii

Coxiella burnetii is the only member of the order Legionellales known to primarily infect vertebrates. The Q fever pathogen is also unusual in that it replicates within an acidified phagolysosome-like vacuole. The evolutionary origins of the virulence determinants underlying this lifestyle remain unclear. More broadly, little is known about how virulence-related traits arise in specialized intracellular lineages, where access to foreign-origin DNA may be more episodic. To address this question, we used Legionellales-wide comparative phylogenomics to reconstruct the gain and loss of traits affecting host interaction, immune evasion, intracellular survival, and metabolism. We found that many virulence-associated traits in C. burnetii predate the modern pathogen and were assembled stepwise in ancestors that likely occupied niches distinct from the acidified vacuolar niche of modern C. burnetii. The common ancestor shared with soft-tick Coxiella endosymbionts likely encoded most C. burnetii type IVB secretion system effectors, indicating that much of the host-manipulation repertoire in C. burnetii was already present before the emergence of the modern pathogen. Distinctive lipopolysaccharide features associated with immune evasion also appear to have accumulated progressively within the Coxiella lineage, including genes implicated in synthesis of virenose, a unique O-antigen sugar critical for C. burnetii virulence. Traits likely to support replication in the acidic Coxiella-containing vacuole likewise accumulated gradually, with generalized stress-tolerance functions predating acquisition of an Mrp cation/proton antiporter that may have further supported pH homeostasis. Additional changes in sugar transport and catabolism, glycolytic control, and respiratory metabolism likely enhanced metabolic flexibility and access to diverse substrates in this nutrient-rich niche. Together, these findings support a model in which vertebrate pathogenicity in C. burnetii emerged through stepwise remodeling of an ancestral host-associated lineage and provide a framework for understanding how virulence-related traits evolve in specialized intracellular pathogens. AUTHOR SUMMARYCoxiella burnetii is the bacterium that causes Q fever, a disease that can spread from animals to humans. Unlike its close relatives, C. burnetii primarily infects vertebrates and grows inside an acidic compartment within host cells. New bacterial pathogens often evolve by gaining genes from other bacteria, but how virulence evolves in lineages that grow only inside host cells, where opportunities to gain new genes may be infrequent, remains unclear. We wanted to understand how C. burnetii evolved the traits needed for its distinctive intracellular lifestyle. By comparing its genome to those of related bacteria across the order Legionellales, we found that features involved in host manipulation, immune evasion, acid tolerance, and nutrient use appeared at different times in its ancestry rather than being acquired all at once by the modern pathogen. Our findings suggest that specialized intracellular pathogens can emerge through gradual changes in ancestral host-associated lineages, including gene acquisition, gene loss, retention of older traits, and repurposing of existing functions.

microbiology↗
bioRxiv2026

Phylogenomics of Cypriniformes, the most diverse order of freshwater fishes: consensus, challenges and limitations

Cypriniformes, the most species-rich order of freshwater fishes ([~]5,000 species), represents a key lineage for understanding vertebrate diversification in freshwater ecosystems. This clade includes several highly miniaturized and understudied lineages whose phylogenetic placements have long remained contentious. Here, we present the first phylogenomic analysis of Cypriniformes with complete family-level representation and broad genus-level coverage, encompassing 316 species comprising approximately 30% of all described genera. Our dataset integrates 257 newly assembled genomes with publicly available resources and analyzes multiple sets of genome-wide markers using both concatenation-based and coalescent-aware approaches. The general concordance among analytical frameworks indicates that the backbone topology of Cypriniformes is now established, allowing clear identification between well-supported clades and regions of persistent conflict. Our results strengthen the evolutionary relationships of several miniaturized lineages, while identifying recalcitrant relationships shaped by both biological processes and model artefacts that can yield superficially similar patterns of phylogenetic conflict. Our study substantially expands genomic representation and establishes a phylogenomic foundation for future comparative, developmental, and evolutionary research in this freshwater radiation.

evolutionary biology↗
bioRxiv2025

Coxiella burnetii strains elicit distinct inflammatory responses in human macrophages

Coxiella burnetii, the causative agent of human Q fever, subverts macrophage antimicrobial functions to establish an intracellular replicative niche. To better understand the host-pathogen interactions, we investigated the transcriptional responses of human alveolar macrophages (hAMs) infected with virulent (NMI, G), attenuated (NMII), and avirulent (Dugway) strains of C. burnetii. RNA sequencing analysis revealed that all strains activated proinflammatory pathways, particularly IL-17 signaling, though the magnitude and nature of the response varied by strain. Dugway infection induced the most robust transcriptional response and consistent M1-like macrophage polarization, while responses to NMI and NMII were more variable. Cytokine assays confirmed significant secretion of effectors downstream of IL-17 signaling, but only at later stages of infection. Single-cell RNA sequencing further revealed heterogeneity in macrophage response to C. burnetii infection, with distinct subpopulations exhibiting divergent inflammatory profiles. These findings highlight the complexity of macrophage responses to C. burnetii and underscore the importance of strain-specific and cell-specific factors in shaping host immunity. Understanding these dynamics may inform the development of targeted therapies for Q fever.

microbiology↗
bioRxiv2025

Engineering Sustained-Release Broadly Neutralizing Antibody Formulations

Sustained serum levels of broadly neutralizing antibodies (bnAbs) are crucial for effective passive immunization against infectious diseases as protection persists only while these bnAbs remain at adequate concentrations within the body. Current obstacles, such as poor pharmacokinetics (PK) and burdensome administration, must be overcome to make bnAbs a viable option for pre- and post-exposure prophylaxis. In this work, we explore how a polymer-nanoparticle (PNP) hydrogel depot technology can be engineered to prolong protein delivery. In-vivo studies in mice and rats demonstrate prolonged protein release, and modeling efforts predict the impact of both the elimination half-life of the active pharmaceutical ingredient and hydrogel depot volume on overall pharmacokinetics. Moreover, flow cytometry characterization reveals that immune cell infiltration into the hydrogel depot can result in faster-than-expected release of antibody cargo on account of active transport via cellular uptake. We then demonstrate that co-formulation of antibodies with an anti-inflammatory agent reduces cellular infiltration and resulting active transport, further extending delivery and pharmacokinetics. Finally, multicompartmental modeling predicts the human PK profiles of clinically relevant HIV bnAbs delivered via subcutaneous hydrogel injection. These findings aid in the development of next generation hydrogel materials that stabilize and slowly release bnAbs for long-term pre-exposure immunoprophylaxis.

bioengineering↗
bioRxiv2025

Genomic signatures of terrestrial adaptation in air-breathing catfishes (Clariidae)

Air-breathing catfishes of the family Clariidae exhibit extraordinary adaptations that enable them to survive outside water for extended periods, yet the genetic and genomic basis of these adaptations remain poorly understood. To study these adaptations, we sequenced and assembled two high-quality genomes of two clariid species, Clarias gariepinus and Clarias dussumieri and compare them with previously available genomes of 23 catfish species across nine families. By reconstructing the whole-genome phylogeny and examining patterns of positive selection and gene family evolution, we found unique signatures associated with terrestrial adaptation in clariids. Our analysis revealed that a high proportion of genes were positively selected in clariids, that play critical roles in hypoxia tolerance, thermoregulation, metabolism, and DNA repair, which are key traits for terrestrial adaptation. Additionally, we observed significant expansions in gene families, including Myoglobin (involved in oxygen transport), immunity-related genes, and xenobiotic degradation pathways, highlighting their importance in environmental resilience and detoxification. Together, these findings provide a comprehensive understanding of the genomic changes facilitating the terrestrial adaptation of clariids. This study also highlights the contribution of genome evolution to their resilience, adaptability to novel environments, and invasiveness, offering valuable insights into the genetic basis of ecological niche diversification.

evolutionary biology↗
bioRxiv2025

Transfusion of allogenic murine HOD red blood cells preferentially induces low-affinity, short-lived IgG antibodies that are germinal center independent

Transfusion-induced anti-red blood cell (RBC) alloantibodies pose a significant risk to patients who require chronic transfusions. Anti-RBC alloantibodies can be remarkably short-lived (i.e. evanescent), leading to clinically relevant alloantibodies that are not detected in later pre-transfusion antibody screens. Subsequent transfusion of alloantigen-positive RBCs stimulates a rapid memory antibody response that may induce a delayed hemolytic transfusion reaction (DHTR), causing morbidity and occasional mortality in chronically transfused patients. It is unclear why transfusions favor evanescent antibody responses over long-lived antibodies typically observed upon infections and vaccinations. We therefore turned to the HOD mouse model of RBC alloimmunization to elucidate regulators of antibody persistence in response to allogenic transfusions. By following antibody responses over time in transfused mice, we found that HOD-specific alloantibodies rapidly decay within three months while vaccination-induced antibodies remain constant. Thus, the HOD model recapitulates RBC antibody evanescence. The rapid antibody evanescence suggests that transfusion is a poor inducer of germinal centers (GCs), specialized immunological structures where B cells differentiate into germinal center B (GC B) cells and undergo iterative rounds of affinity maturation, ultimately differentiating into long-lived plasma cells that can produce antibodies for decades. Consistent with this hypothesis, we failed to observe an increase in GC B cell formation in response to transfusion, and the majority of anti-RBC alloantibodies were low affinity when compared to vaccination. To formally test the functional requirement for GCs in anti-RBC alloantibody production, we employed two orthogonal approaches to disrupt GC formation: i) day 4 CD40L blockade and ii) genetic disruption of the GC-transcription factor BCL6 selectively in B cells. Both approaches fully blocked GC formation, yet anti-RBC alloantibody production was unchanged. Collectively, our data demonstrate that anti-HOD RBC alloantibodies are GC-independent, low affinity and short-lived. The GC-independence of HOD RBC IgG responses has important implications for understanding the cellular and molecular pathways that regulate the humoral immune response to transfused RBCs, potentially explaining anti-RBC alloantibody evanescence patterns in patients.

immunology↗
bioRxiv2024

Activation of locus coeruleus noradrenergic neuronsrapidly drives homeostatic sleep pressure

Homeostatic sleep regulation is essential for optimizing the amount and timing of sleep, but the underlying mechanism remains unclear. Optogenetic activation of locus coeruleus noradrenergic neurons immediately increased sleep propensity following transient wakefulness. Fiber photometry showed that repeated optogenetic or sensory stimulation caused rapid declines of locus coeruleus calcium activity and noradrenaline release. This suggests that functional fatigue of noradrenergic neurons, which reduces their wake-promoting capacity, contributes to sleep pressure.

neuroscience↗
bioRxiv2023

The adhesion GPCRs CELSR1-3 and LPHN3 engage G proteins via distinct activation mechanisms

Adhesion GPCRs (aGPCRs) are a large GPCR class that direct diverse fundamental biological processes. One prominent mechanism for aGPCR agonism involves autoproteolytic cleavage, which generates an activating, membrane-proximal tethered agonist (TA). How universal this mechanism is for all aGPCRs is unclear. Here, we investigate G protein induction principles of aGPCRs using mammalian LPHN3 and CELSR1-3, members of two aGPCR families conserved from invertebrates to vertebrates. LPHNs and CELSRs mediate fundamental aspects of brain development, yet CELSR signaling mechanisms are unknown. We found that CELSR1 and CELSR3 are cleavage-deficient, while CELSR2 is efficiently cleaved. Despite differential autoproteolysis, CELSR1-3 all engage GS, and CELSR1 or CELSR3 TA point mutants retain GS coupling activity. CELSR2 autoproteolysis enhances GS coupling, yet acute TA exposure alone is insufficient. These studies support that aGPCRs signal via multiple paradigms and provide insights into CELSR biological function.

cell biology↗
bioRxiv2022

LIQ HD (Lick Instance Quantifier Home cage Device): An open-source tool for recording undisturbed two-bottle drinking behavior in a home cage environment

Investigation of rodent drinking behavior has provided insight into drivers of thirst, circadian rhythms, anhedonia, and drug and ethanol consumption. Traditional methods of recording fluid intake involve weighing bottles, which is cumbersome and lacks temporal resolution. Several open-source devices have been designed to improve drink monitoring, particularly for two-bottle choice tasks. However, recent designs are limited by the use of infrared photobeam sensors and incompatibility with prolonged undisturbed use in ventilated home cages. Beam-break sensors lack accuracy for bout microstructure analysis and are prone to damage from rodents. Thus, we designed LIQ HD (Lick Instance Quantifier Home cage Device) with the goal of utilizing capacitive sensors to increase accuracy and analyze lick microstructure, building a device compatible with ventilated home cages, increasing scale with prolonged undisturbed recordings, and creating a design that is easy to build and use with an intuitive touchscreen graphical user interface. The system tracks two-bottle choice licking behavior in up to 18 rodent cages, or 36 single bottles, on a minute-to-minute timescale controlled by a single Arduino microcontroller. The data are logged to a single SD card, allowing for efficient downstream analysis. With sucrose, quinine, and ethanol two-bottle choice tasks, we validated that LIQ HD has superior accuracy compared to photobeam sensors. The system measures preference over time and changes in bout microstructure, with undisturbed recordings lasting up to 7 days. All designs and software are open-source to allow other researchers to build upon the system and adapt LIQ HD to their animal home cages. Significance StatementTwo-bottle choice drinking tasks are traditionally performed by periodically weighing bottles, which is cumbersome and lacks temporal resolution. Several open-source tools have been developed to improve drink monitoring in various settings. However, no open-source devices have been designed specifically to investigate temporally precise two-bottle choice drinking behavior and bout microstructure during prolonged undisturbed tasks in mouse ventilated home cages at a large scale. Our design, LIQ HD (Lick Instance Quantifier Home cage Device), is a home cage compatible system that utilizes capacitive sensors for highly accurate lick detection during two-bottle choice tasks in up to 18 cages driven by a single Arduino microcontroller. The system is low-cost, easy to build, and controlled via touchscreen with an intuitive graphical user interface.

neuroscience↗
bioRxiv2022

MicroRNAs contribute to the host response to Coxiella burnetii

MicroRNAs (miRNAs), a class of small non-coding RNAs, are critical to gene regulation in eukaryotes. They are involved in modulating a variety of physiological processes, including the host response to intracellular infections. Little is known about miRNA functions during infection by Coxiella burnetii, the causative agent of human Q fever. This bacterial pathogen establishes a large replicative vacuole within macrophages by manipulating host processes such as apoptosis and autophagy. We investigated miRNA expression in C. burnetii-infected macrophages and identified several miRNAs that were down- or up-regulated during infection. We further explored the functions of miR-143-3p, an miRNA whose expression is down-regulated in macrophages infected with C. burnetii, and show that increasing the abundance of this miRNA in human cells results in increased apoptosis and reduced autophagy - conditions that are unfavorable to C. burnetii intracellular growth. In sum, this study demonstrates that C. burnetii infection elicits a robust miRNA-based host response, and because miR-143-3p promotes apoptosis and inhibits autophagy, down-regulation of miR-143-3p expression during C. burnetii infection likely benefits the pathogen.

microbiology↗
bioRxiv2021

Genome-wide identification of novel sRNAs in Streptococcus mutans

Streptococcus mutans is a major pathobiont involved in the development of dental caries. Its ability to utilize numerous sugars and to effectively respond to environmental stress promotes S. mutans proliferation in oral biofilms. Because of their quick action and low energetic cost, non-coding small RNAs (sRNAs) represent an ideal mode of gene regulation in stress response networks, yet their roles in oral pathogens have remained largely unexplored. We identified 15 novel sRNAs in S. mutans and show that they respond to four stress-inducing conditions commonly encountered by the pathogen in human mouth: sugar-phosphate stress, hydrogen peroxide exposure, high temperature, and low pH. To better understand the role of sRNAs in S. mutans, we further explored the function of the novel sRNA, SmsR4. Our data demonstrate that SmsR4 regulates the EIIA component of the sorbitol phosphotransferase system, which transports and phosphorylates the sugar alcohol sorbitol. The fine-tuning of EIIA availability by SmsR4 likely promotes S. mutans growth while using sorbitol as the main carbon source. Our work lays a foundation for understanding the role of sRNAs in regulating gene expression in stress response networks in S. mutans and highlights the importance of the underexplored phenomenon of posttranscriptional gene regulation in oral bacteria. IMPORTANCESmall RNAs (sRNAs) are important gene regulators in bacteria, but the identities and functions of sRNAs in Streptococcus mutans, the principal bacterium involved in the formation of dental caries, are unknown. In this study, we identified 15 putative sRNAs in S. mutans and show that they respond to four common stress-inducing conditions present in human mouth: sugar-phosphate stress, hydrogen peroxide exposure, high temperature, and low pH. We further show that the novel sRNA SmsR4 likely modulates sorbitol transport into the cell by regulating SMU_313 mRNA, which encodes the EIIA subunit of the sorbitol phosphotransferase system. Gaining a better understanding of sRNA-based gene regulation may provide new opportunities to develop specific inhibitors of S. mutans growth, thereby improving oral health.

microbiology↗
bioRxiv2021

A peroxide-responding sRNA evolved from a peroxidase mRNA

Small RNAs (sRNAs) are critical regulators of gene expression in bacteria, but we lack a clear understanding of how new sRNAs originate and get integrated into regulatory networks. A major obstacle to elucidating their evolution is the difficulty in tracing sRNAs across large phylogenetic distances. To overcome this roadblock, we investigated the prevalence of sRNAs in more than a thousand genomes across Enterobacterales, a bacterial order with a rare confluence of factors that allows robust genome-scale sRNA analyses: several well-studied organisms with fairly conserved genome structures, an established phylogeny, and substantial nucleotide diversity within a narrow evolutionary space. Using a covariance modeling-based approach, we analyzed the presence of hundreds of sRNAs and discovered that a majority of sRNAs arose recently, and uncovered protein-coding genes as a potential source for the generation of new sRNA genes. A detailed investigation of the emergence of OxyS, a peroxide-responding sRNA, demonstrated that it evolved from a 3' end fragment of a peroxidase mRNA. Collectively, our data show that the erosion of protein-coding genes can result in the formation of new sRNAs that continue to be part of the original proteins regulon. This novel insight provides a fresh framework for understanding how new sRNAs originate and get incorporated into preexisting regulatory networks. AUTHOR SUMMARYSmall RNAs (sRNAs) are important gene regulators in bacteria, but it is unclear how new sRNAs originate and become part of regulatory networks that coordinate bacterial response to environmental stimuli. Here, we show that new sRNAs could arise from protein-coding genes and potentially be incorporated into the ancestral proteins regulatory networks. We illustrate this process by defining the origin of OxyS. This peroxide-responding sRNA evolved from and replaced a peroxidase gene, but continues to be part of the peroxide-response regulon. In sum, we describe the source from which OxyS, one of the most well-studied sRNAs, arose, identify protein-coding genes as a potential raw material from which new sRNAs could emerge, and suggest a novel evolutionary path through which new sRNAs could get incorporated into pre-existing regulatory networks.

microbiology↗
bioRxiv2021

Captivity-induced behaviour and spatial learning abilities in an enigmatic, aquifer-dwelling blind eel, Rakthamichthys digressus

We investigated the impact of captive life on behaviour and learning abilities in an enigmatic, aquifer-dwelling blind eel, Rakthamichthys digressus. Of eight major behavioural traits related to exploration and activity in a novel arena, four were significantly altered by life in captivity. While the startle response upon introduction into the arena and overall swimming away from the walls increased after captivity, inactivity exhibited immediately after the startle and the reaction to an external disturbance decreased. We also observed behavioural syndromes between startle responses and horizontal wall following, and between overall activity and vertical wall following; however, these behavioural syndromes were not altered by maintenance in captivity. Interestingly, this blind-eel failed to learn a simple spatial task in a Y-maze apparatus. Captive-associated behavioural changes in R. digressus may influence their survival after reintroduction into natural habitats, and such changes must be taken into account while developing protocols for ex-situ conservation and subsequent release.

animal behavior and cognition↗
bioRxiv2020

Ticks convert pathogenic Coxiella into endosymbionts

Both symbiotic and pathogenic bacteria in the family Coxiellaceae cause morbidity and mortality in humans and animals. For instance, Coxiella-like endosymbionts (CLEs) improve the reproductive success of ticks -- a major disease vector, while Coxiella burnetii is the etiological agent of human Q fever and uncharacterized coxiellae cause infections in both animals and humans. To better understand the evolution of pathogenesis and symbiosis in this group of intracellular bacteria, we sequenced the genome of a CLE present in the soft tick Ornithodoros amblus (CLEOA) and compared it to the genomes of other bacteria in the order Legionellales. Our analyses confirmed that CLEOA is more closely related to C. burnetii, the human pathogen, than to CLEs in hard ticks, and showed that most clades of CLEs contain both endosymbionts and pathogens, indicating that several CLE lineages have evolved independently from pathogenic Coxiella. We also determined that the last common ancestor of CLEOA and C. burnetii was equipped to infect macrophages, and that even though HGT contributed significantly to the evolution of C. burnetii, most acquisition events occurred primarily in ancestors predating the CLEOA-C. burnetii divergence. These discoveries clarify the evolution of C. burnetii, which previously was assumed to have emerged when an avirulent tick endosymbiont recently gained virulence factors via HGT. Finally, we identified several metabolic pathways, including heme biosynthesis, that are likely critical to the intracellular growth of the human pathogen but not the tick symbiont, and show that the use of heme analogs is a promising approach to controlling C. burnetii infections.

microbiology↗
bioRxiv2020

Novel small RNAs expressed by Bartonella bacilliformis under multiple conditions reveal potential mechanisms for persistence in the sand fly vector and human host

Bartonella bacilliformis, the etiological agent of Carrions disease, is a Gram-negative, facultative intracellular alphaproteobacterium. Carrions disease is an emerging but neglected tropical illness endemic to Peru, Colombia, and Ecuador. B. bacilliformis is spread between humans through the bite of female phlebotomine sand flies. As a result, the pathogen encounters significant and repeated environmental shifts during its life cycle, including changes in pH and temperature. In most bacteria, small non-coding RNAs (sRNAs) serve as effectors that may post-transcriptionally regulate the stress response to such changes. However, sRNAs have not been characterized in B. bacilliformis, to date. We therefore performed total RNA-sequencing analyses on B. bacilliformis grown in vitro then shifted to one of ten distinct conditions that simulate various environments encountered by the pathogen during its life cycle. From this, we identified 160 sRNAs significantly expressed under at least one of the conditions tested. sRNAs included the highly-conserved tmRNA, 6S RNA, RNase P RNA component, SRP RNA component, ffH leader RNA, and the alphaproteobacterial sRNAs r45 and speF leader RNA. In addition, 153 other potential sRNAs of unknown function were discovered. Northern blot analysis was used to confirm the expression of eight novel sRNAs. We also characterized a Bartonella bacilliformis group I intron (BbgpI) that disrupts an un-annotated tRNACCUArg gene and determined that the intron splices in vivo and self-splices in vitro. Furthermore, we demonstrated the molecular targeting of Bartonella bacilliformis small RNA 9(BbsR9) to transcripts of the ftsH, nuoF, and gcvT genes, in vitro. Author summaryB. bacilliformis is a bacterial pathogen that is transmitted between humans by phlebotomine sand flies. Bacteria often express sRNAs to fine-tune the production of proteins involved in a wide array of biological processes. We cultured B. bacilliformis in vitro under standard conditions then shifted the pathogen for a period of time to ten distinct environments, including multiple temperatures, pH levels, and infections of human blood and human vascular endothelial cells. After RNA-sequencing, a manual transcriptome search identified 160 putative sRNAs, including seven highly-conserved sRNAs and 153 novel potential sRNAs. We then characterized two of the novel sRNAs, BbgpI and BbsR9. BbgpI is a group I intron (ribozyme) that self-splices and disrupts an unannotated gene coding for a transfer RNA (tRNACCUArg). BbsR9 is an intergenic sRNA expressed under conditions that simulate the sand fly. We found that BbsR9 targets transcripts of the ftsH, nuoF, and gcvT genes. Furthermore, we determined the specific sRNA-mRNA interactions responsible for BbsR9 binding to its target mRNAs through in vitro mutagenesis and binding assays.

microbiology↗
bioRxiv2020

The identity and distribution of Bhavania annandalei Hora 1920 (Cypriniformes: Balitoridae), a hillstream loach endemic to the Western Ghats of India

Bhavania annandalei Hora 1920, is resurrected from the synonymy of B. australis (Jerdon 1849) based on examination of freshly collected topotypic specimens. The two species can be distinguished by a combination of morphological, morphometric and meristic characters, and by genetic distance using mitochondrial cox1 gene. The distribution of B. annandalei is restricted to the river systems draining the Agasthyamalai hills, below the Shencottah Gap in southern Western Ghats.Competing Interest StatementThe authors have declared no competing interest.View Full Text

zoology↗
bioRxiv2019

Coxiella burnetii small RNA 12 binds CsrA regulatory protein and transcripts for the CvpD type IV effector, regulates pyrimidine and methionine metabolism, and is necessary for optimal intracellular growth and vacuole formation during infection

Coxiella burnetii is an obligate intracellular gammaproteobacterium and zoonotic agent of Q fever. We previously identified 15 small non-coding RNAs (sRNAs) of C. burnetii. One of them, named CbsR12 (Coxiella burnetii small RNA 12) is highly expressed during growth in axenic medium and becomes even more dominant during infection of cultured mammalian cells. Secondary structure predictions of CbsR12 revealed four putative CsrA-binding sites in single-stranded segments of stem loops with consensus AGGA/ANGGA motifs. From this foundation, we determined that CbsR12 binds to recombinant C. burnetii CsrA-2, but not CsrA-1, proteins in vitro. Moreover, through a combination of in vitro and in vivo assays, we identified several in trans mRNA targets of CbsR12. Of these, we determined that CbsR12 binds to and upregulates translation of carA transcripts coding for carbamoyl phosphate synthetase A; an enzyme that catalyzes the first step of pyrimidine biosynthesis. In addition, CbsR12 binds and downregulates translation of metK transcripts coding for S-adenosyl methionine (SAM) synthase, a component of the methionine cycle. Furthermore, we found that CbsR12 binds to and downregulates the quantity of cvpD transcripts, coding for a type IVB effector protein, in vitro and in vivo. Finally, we found that CbsR12 is necessary for full expansion of Coxiella-containing vacuoles (CCVs) and affects bacterial growth rates in a dose-dependent manner in the early phase of infecting THP-1 cells. This is the first detailed characterization of a trans-acting sRNA of C. burnetii and the first example of a bacterial sRNA that regulates both CarA and MetK expression. CbsR12 is also one of only a few identified trans-acting sRNAs that interacts with CsrA. Results illustrate the importance of sRNA-mediated regulation in establishment of the intracellular CCV niche.\n\nAuthor summaryC. burnetii is an obligate intracellular bacterial pathogen that is transmitted to humans from animal reservoirs. Upon inhalation of aerosolized C. burnetii, the agent is phagocytosed by macrophages in the lung. The pathogen subverts macrophage-mediated degradation and resides in a large, intracellular, acidic vacuole, termed the Coxiella-containing vacuole (CCV). Small RNAs (sRNAs) are not translated into proteins. Instead, they target mRNAs in order to up- or down-regulate their stability and translation. Alternatively, some sRNAs bind to regulatory proteins and serve as \"sponges\" that effectively sequester the proteins and inhibit their function. C. burnetiis CbsR12 sRNA is highly expressed during infection in order to expand the CCV, and it works by a variety of mechanisms, including: 1) directly regulating transcripts of several metabolic genes that aid in bacterial replication, 2) binding to and regulating transcripts of a type IV effector protein that aids in infection, and 3) indirectly regulating an unknown number of genes by binding to a homolog of the global regulatory protein, CsrA. CbsR12 represents one of only a few sRNAs known to bind and sequester CsrA while also directly regulating mRNAs.

microbiology↗