James E. Samuel

Coxiella burnetii (Cb), the causative agent of Q fever, replicates within host macrophages by modulating innate immune responses through its type IVB secretion system (T4SS). Host cells sense pathogens via Pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), to recognize pathogen-associated molecular patterns (PAMPs) and initiate signaling pathways that drive pro-inflammatory cytokine and interferon responses. Toll-like receptor 3 (TLR3) triggers the nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Interferon regulatory factors (IRFs) resulting in pro-inflammatory cytokine production and type I interferon (IFN-I) induction. Here we demonstrate that Cb requires T4SS to suppress TLR3-induced NF-κB and IRF transcriptional responses. Furthermore, RNA purified from both virulent and avirulent Cb is sufficient to activate TLR3, identifying pathogen RNA as a relevant PAMP in this context. Using a pulmonary infection model with virulent Cb, we found that TLR3/ Toll-Interleukin-1 Receptor Domain-Containing Adapter Protein Inducing Interferon Beta (TRIF)-dependent innate immune pathway inhibits cachexia. In contrast, signaling through the interferon-α/β receptor (IFNAR) restricts bacterial dissemination, indicating that these pathways play distinct but complementary roles in host defense. Mechanistically, Cb suppresses TLR3/TRIF signaling in a T4SS-dependent manner by preventing host TLR3 recruitment to the Coxiella-containing vacuole (CCV) and disrupting TRIF–TNF Receptor-Associated Factor 6 (TRAF6) interactions, thereby selectively inhibiting impairing NF-κB activation. Consistent with this, we identify five T4SS effector proteins that attenuate TLR3-induced NF-κB signaling, including CBU1292, which suppresses both NF-κB and IRF-dependent responses downstream of TLR3. Author summary: Pathogenic bacteria have evolved mechanisms to evade or manipulate host immune defenses. Coxiella burnetii (Cb), the causative agent of Q fever, replicates within immune cells and interferes with innate immune signaling pathways. We found that its type IVB secretion system (T4SS) express effectors that inhibit immune responses triggered by Toll-like receptor 3 (TLR3), a sensor that detects microbial RNA. Specifically, Cb suppresses signaling through the adaptor protein TRIF, thereby reducing activation of key immune regulators, NF-κB and interferon regulatory factors (IRFs), which are required for inflammatory and antiviral responses. Using a mouse model of lung infection, we found that disruption of TLR3/TRIF signaling and type I interferon receptor (IFNAR) signaling leads to different disease outcomes, indicating that these pathways play distinct roles in controlling infection and disease severity. We also identified several Cb-secreted effectors that interfere with TLR3-mediated immune activation, including one that inhibits both NF-κB and interferon responses. Together, our findings reveal how Cb evades immune detection by targeting a critical RNA-sensing pathway and highlight distinct host defense mechanisms that limit bacterial infection and disease.

C. burnetii (Cb) is an obligate intracellular bacterial pathogen that replicates within alveolar macrophages following aerosol infection. Unlike most intracellular bacteria, Cb establishes a lysosome-derived replicative niche (Coxiella-containing vacuole or CCV) through the action of its Type IVB secretion system (T4BSS). This system translocates a large repertoire of effector proteins into the host cytoplasm after phagosome acidification. These effectors interfere with diverse signaling pathways to co-opt host processes, such as vesicle trafficking, ubiquitylation, gene expression and lipid metabolism, promoting pathogen survival without triggering robust proinflammatory signaling or host cell death pathways. This effector-triggered immune silencing is particularly unique given the central role of macrophages as innate immune sentinels. In this review, we examine Cb T4BSS effectors that have been characterized as central determinants of innate immunity modulation. We discuss innate immune sensing pathways potentially engaged during infection, including Toll-like receptors, NOD-like receptors, RIG-I-like receptors, inflammasomes, and interferon signaling pathways, and highlight evidence indicating that these pathways are actively suppressed. Emphasis is placed on effector-mediated regulation of NF-κB signaling, type I interferon responses, and inflammasome activation. Finally, we address unresolved questions related to effector-triggered immunity, redundancy in immune suppression, and discrepancies between in vitro and in vivo infection models.

Coxiella burnetii (Cb), the causative agent of Q fever, replicates within host macrophages by modulating immune responses through poorly understood mechanisms. Long non-coding RNAs (lncRNAs) are crucial yet underexplored regulators of inflammation, particularly in Cb pathogenesis. Employing a comparative transcriptomic analysis of THP-1 macrophages infected with 16 different microbes, we dissect a core set of immune-responsive lncRNAs such as MAILR, LINC01215, PACER, and MROCKI-common to human anti-pathogen responses, and distinguish them from lncRNAs specifically altered at early (1 h) time points in individual infections. In particular, our approach identifies lncRNA CYP1B1-AS1 as specifically upregulated in a spatiotemporal manner along with CYP1B1 in cis during Cb infection. Promoter assays confirm their co-regulation via a shared bidirectional promoter, while aryl hydrocarbon receptor (AHR)-lucia luciferase and nuclear translocation assays demonstrate that Cb infection activates AHR, driving their transcription. Knockdown of CYP1B1-AS1 or CYP1B1 alone disrupts mitochondrial homeostasis, increases ROS and mitochondrial dysfunction, and exacerbates apoptosis during infection. These findings position the CYP1B1-AS1/CYP1B1 axis as a key regulator of mitochondrial homeostasis under AHR signaling, supporting an intracellular environment that benefits Cb replication. Our results highlight the critical roles of lncRNAs in immune regulation and provide a valuable resource for future lncRNA research.

Uncultivated but abundant soil microorganisms have untapped potential for producing broad ranges of natural products, as well as for bioremediation. However, cultivating soil microorganisms while maintaining a broad microorganism diversity to enable phenotyping and functional analysis of as diverse individual isolates as possible remains challenging. In this study, we developed and tested the ability of several culture media formulations that contain defined soil metabolites or soil extracts to maintain microorganism diversity during culture. We also assessed their performance in microfluidic droplet cultivation where single-soil microorganism isolates were encapsulated and cultivated in picoliter-volume water-in-oil emulsion droplets to enable clonal growth needed for downstream functional analyses. Our results show that droplet cultivation with media supplemented by soil extract or soil metabolites enables the recovery of soil microorganisms with higher diversity (up to 1.5-fold higher richness) compared to bulk cultivation methods. Importantly, 1.7-fold more of less abundant (in situ soil microorganism community in microfluidic droplets and conducting in-droplet cultivation in media supplemented by soil extract or soil metabolites. This method enables the recovery of a broader diversity of the original microorganism community, laying the groundwork for a high-throughput phenotyping of these diverse microorganisms from their natural habitats.

Coxiella burnetii(Cb), the causative agent of Q fever, replicates within host macrophages by modulating immune responses through poorly understood mechanisms. Long non-coding RNAs (lncRNAs) are emerging as critical regulators of inflammation, yet their role in Cb pathogenesis remains largely unexplored. Here, we employed a global transcriptomic approach to identify lncRNAs specific to Cb infection in THP-1 derived macrophages, compared to 15 other microbial infections. CYP1B1-AS1 was uniquely regulated in a spatio-temporal manner during Cb infection. Promoter assays revealed that CYP1B1-AS1 is transcribed by AHR from a bidirectional promoter, enhancing CYP1B1 expression in cis. Inhibition of CYP1B1-AS1 and CYP1B1 increased reactive oxygen species (ROS), mitochondrial membrane depolarization, and apoptosis, suggesting their role in dampening host cell death. Additionally, immunoprecipitation followed by mass spectrometry identified the mitochondrially localized Cb effector CBU_0937 as an interactor of the CYP1B1 enzyme. These events facilitate Cb intracellular survival. Our findings identify CYP1B1-AS1 as a potential molecular target for combating Cb infection.

Local and systemic reactogenic responses to Q-VAX have prevented licensing of this vaccine outside of Australia. These reactogenic responses occur in previously sensitized individuals and have not been well defined at the cellular level, in part because many studies have been done in guinea pigs that have limited molecular tools. We previously characterized a mouse model of reactogenicity where local reaction sites showed an influx of CD8+ and IFNγ-expressing IL17a+ CD4+ T cells consistent with a Th1 delayed-type hypersensitivity. In this study, we determined, using depletion and adoptive transfer experiments, that both anti- Coxiella antibodies and CD4+ T cells were essential for localized reactions at the site of vaccination. Furthermore, IFNγ depletion showed significant histological changes at the local reaction sites demonstrating the essential nature of this cytokine to reactogenicity. In addition to the cells and cytokines required for this response, we determined that whole cell vaccine (WCV) material remained at the site of vaccination for at least 26 weeks post-injection. Transmission electron microscopy (TEM) of these sites demonstrated intact rod-shaped bacteria at 2 weeks post-injection and partially degraded bacteria within macrophages at 26 weeks post-injection. Finally, because small cell variants (SCVs) are an environmentally stable form, we determined that local reactions were more severe when the WCV material was prepared with higher levels of SCVs compared to typical WCV or with higher levels of large cell variant (LCV). These studies support the hypothesis that antigen persistence at the site of injection contributes to this reactogenicity and that anti- Coxiella antibodies, CD4+ T cells, and IFNγ each contribute to this process.

Local and systemic reactogenic responses to Q-VAX® have prevented licensing of this vaccine outside of Australia. These reactogenic responses occur in previously sensitize individuals and have not been well defined at the cellular level, in part because many studies have been done in guinea pigs that have limited molecular tools. We previously characterized a mouse model of reactogenicity where local reactions sites showed an influx of CD8+ and IFNγ-expressing IL17a+ CD4+ T cells consistent with a Th1 delayed-type hypersensitivity. In this study we determined using depletion and adoptive transfer experiments that both anti- Coxiella antibodies and CD4+ T cells were essential for localized reactions at the site of vaccination. Furthermore, IFNγ depletion showed significant histological changes at the local reaction sites demonstrating the essential nature of this cytokine to reactogenicity. In addition to the cells and cytokines required for this response, we determined WCV material remained at the site of vaccination for at least 26 weeks post-injection. Transmission electron microscopy of these sites demonstrated intact rod-shaped bacteria at 2 weeks post-injection and partially degraded bacteria within macrophages at 26 weeks post-injection. Finally, since SCVs are an environmentally stable form, we determined that local reactions were more severe when the WCV material was prepared with higher levels of SCVs compared to typical WCV or with higher levels of LCV. These studies support the hypothesis that antigen persistence at the site of injection contributes to this reactogenicity and that anti- Coxiella antibodies, CD4+ T cells, and IFNγ each contribute to this process.

Obligate intracellular bacteria have remained those for which effective vaccines are unavailable, mostly because protection does not solely rely on an antibody response. Effective antibody-based vaccines, however, have been developed against extracellular bacteria pathogens or toxins. Additionally, obligate intracellular bacteria have evolved many mechanisms to subvert the immune response, making vaccine development complex. Much of what we know about protective immunity for these pathogens has been determined using infection-resolved cases and animal models that mimic disease. These studies have laid the groundwork for antigen discovery, which, combined with recent advances in vaccinology, should allow for the development of safe and efficacious vaccines. Successful vaccines against obligate intracellular bacteria should elicit potent T cell memory responses, in addition to humoral responses. Furthermore, they ought to be designed to specifically induce strong cytotoxic CD8+ T cell responses for protective immunity. This review will describe what we know about the potentially protective immune responses to this group of bacteria. Additionally, we will argue that the novel delivery platforms used during the Sars-CoV-2 pandemic should be excellent candidates to produce protective immunity once antigens are discovered. We will then look more specifically into the vaccine development for Rickettsiaceae, Coxiella burnetti, and Anaplasmataceae from infancy until today. We have not included Chlamydia trachomatis in this review because of the many vaccine related reviews that have been written in recent years.

The long non-coding RNAs (lncRNAs) are evolutionarily conserved classes of non-coding regulatory transcripts of > 200 nucleotides in length. They modulate several transcriptional and post-transcriptional events in the organism. Depending on their cellular localization and interactions, they regulate chromatin function and assembly; and alter the stability and translation of cytoplasmic mRNAs. Although their proposed range of functionality remains controversial, there is increasing research evidence that lncRNAs play a regulatory role in the activation, differentiation and development of immune signaling cascades; microbiome development; and in diseases such as neuronal and cardiovascular disorders; cancer; and pathogenic infections. This review discusses the functional roles of different lncRNAs in regulation of host immune responses, signaling pathways during host-microbe interaction and infection caused by obligate intracellular bacterial pathogens. The study of lncRNAs is assuming significance as it could be exploited for development of alternative therapeutic strategies for the treatment of severe and chronic pathogenic infections caused by Mycobacterium, Chlamydia and Rickettsia infections, as well as commensal colonization. Finally, this review summarizes the translational potential of lncRNA research in development of diagnostic and prognostic tools for human diseases.