Coxiella burnetii inhibits nuclear translocation of TFEB, the master transcription factor for lysosomal biogenesis.

Coxiella burnetii is a highly infectious, Gram-negative, obligate intracellular bacterium and the causative agent of human Q fever. The Coxiella Containing Vacuole (CCV) is a modified phagolysosome that forms through fusion with host endosomes and lysosomes. While an initial acidic pH < 4.7 is essential to activate Coxiella metabolism, the mature, growth-permissive CCV has a luminal pH of ~5.2 that remains stable throughout infection. Inducing CCV acidification to a lysosomal pH (~4.7) causes Coxiella degradation, suggesting that Coxiella regulates CCV pH. Supporting this hypothesis, Coxiella blocks host lysosomal biogenesis, leading to fewer host lysosomes available to fuse with the CCV. Host cell lysosome biogenesis is primarily controlled by the transcription factor EB (TFEB), which binds Coordinated Lysosomal Expression And Regulation (CLEAR) motifs upstream of genes involved in lysosomal biogenesis and function. TFEB is a member of the microphthalmia/transcription factor E (MiT/TFE) protein family, which also includes MITF, TFE3, and TFEC. This study examines the roles of MiT/TFE proteins during Coxiella infection. We found that in cells lacking TFEB, both Coxiella growth and CCV size increase. Conversely, TFEB overexpression or expression in the absence of other family members leads to significantly less bacterial growth and smaller CCVs. TFE3 and MITF do not appear to play a significant role during Coxiella infection. Surprisingly, we found that Coxiella actively blocks TFEB nuclear translocation in a Type IV Secretion System-dependent manner, thus decreasing lysosomal biogenesis. Together, these results suggest that Coxiella inhibits TFEB nuclear translocation to limit lysosomal biogenesis, thus avoiding further CCV acidification through CCV-lysosomal fusion. IMPORTANCE: The obligate intracellular bacterial pathogen Coxiella burnetii causes the zoonotic disease Q fever, which is characterized by a debilitating flu-like illness in acute cases and life-threatening endocarditis in patients with chronic disease. While Coxiella survives in a unique lysosome-like vacuole called the Coxiella Containing Vacuole (CCV), the bacterium inhibits lysosome biogenesis as a mechanism to avoid increased CCV acidification. Our results establish that transcription factor EB (TFEB), a member of the microphthalmia/transcription factor E (MiT/TFE) family of transcription factors that regulate lysosomal gene expression, restricts Coxiella infection. Surprisingly, Coxiella blocks TFEB translocation from the cytoplasm to the nucleus, thus downregulating the expression of lysosomal genes. These findings reveal a novel bacterial mechanism to regulate lysosomal biogenesis.

Drosophila melanogaster Sting mediates Coxiella burnetii infection by reducing accumulation of reactive oxygen species.

The Gram-negative bacterium Coxiella burnetii is the causative agent of query fever in humans and coxiellosis in livestock. C. burnetii infects a variety of cell types, tissues, and animal species including mammals and arthropods, but there is much left to be understood about the molecular mechanisms at play during infection in distinct species. Human stimulator of interferon genes (STING) induces an innate immune response through the induction of type I interferons (IFNs), and IFN promotes or suppresses C. burnetii replication, depending on tissue type. Drosophila melanogaster contains a functional STING ortholog (Sting) which activates NF-κB signaling and autophagy. Here, we sought to address the role of D. melanogaster Sting during C. burnetii infection to uncover how Sting regulates C. burnetii infection in flies. We show that Sting-null flies exhibit higher mortality and reduced induction of antimicrobial peptides following C. burnetii infection compared to control flies. Additionally, Sting-null flies induce lower levels of oxidative stress genes during infection, but the provision of N-acetyl-cysteine (NAC) in food rescues Sting-null host survival. Lastly, we find that reactive oxygen species levels during C. burnetii infection are higher in Drosophila S2 cells knocked down for Sting compared to control cells. Our results show that at the host level, NAC provides protection against C. burnetii infection in the absence of Sting, thus establishing a role for Sting in protection against oxidative stress during C. burnetii infection.

Metabolism and physiology of pathogenic bacterial obligate intracellular parasites.

Bacterial obligate intracellular parasites (BOIPs) represent an exclusive group of bacterial pathogens that all depend on invasion of a eukaryotic host cell to reproduce. BOIPs are characterized by extensive adaptation to their respective replication niches, regardless of whether they replicate within the host cell cytoplasm or within specialized replication vacuoles. Genome reduction is also a hallmark of BOIPs that likely reflects streamlining of metabolic processes to reduce the need for de novo biosynthesis of energetically costly metabolic intermediates. Despite shared characteristics in lifestyle, BOIPs show considerable diversity in nutrient requirements, metabolic capabilities, and general physiology. In this review, we compare metabolic and physiological processes of prominent pathogenic BOIPs with special emphasis on carbon, energy, and amino acid metabolism. Recent advances are discussed in the context of historical views and opportunities for discovery.

The chlamydial transcriptional regulator Euo is a key switch in cell form developmental progression but is not involved in the committed step to the formation of the infectious form.

Bacteria in the genus Chlamydia are a significant health burden worldwide. They infect a wide range of vertebrate animals, including humans and domesticated animals. In humans, C. psittaci can cause zoonotic pneumonia, while C. pneumoniae causes a variety of respiratory infections. Infections with C. trachomatis cause ocular or genital infections. All chlamydial species are obligate intracellular bacteria that replicate exclusively inside of eukaryotic host cells. Chlamydial infections are dependent on a complex infection cycle that depends on transitions between specific cell forms. This cycle consists of cell forms specialized for host cell invasion, the elementary body (EB), and a form specialized for intracellular replication, the reticulate body (RB). In addition to the EB and RB, there is a transitionary cell form that mediates the transformation between the RB and the EB, the intermediate body (IB). In this study, we ectopically expressed the regulatory protein Euo and showed that high levels of expression resulted in reversible arrest of the development cycle. The arrested chlamydial cells were trapped phenotypically at an early IB stage of the cycle. These cells had exited the cell cycle but had not shifted gene expression from RB like to IB/EB like. This arrested state was dependent on continued expression of Euo. When ectopic expression was reversed, Euo levels dropped in the arrested cells which led to the repression of native Euo expression and the resumption of the developmental cycle. Our data are consistent with a model where Euo expression levels impact IB maturation to the infectious EB but not the production of the IB form. IMPORTANCE: Bacterial species in the Chlamydiales order infect a variety of vertebrate animals and are a global health concern. They cause various diseases in humans, including genital and respiratory infections. The bacteria are obligate intracellular parasites that rely on a complex infectious cycle involving multiple cell forms. All species share the same life cycle, transitioning through different states to form the infectious elementary body (EB) to spread infections to new hosts. The Euo gene, encoding a DNA-binding protein, is involved in regulating this cycle. This study showed that ectopic expression of Euo halted the cycle at an early stage. This arrest depended on continued Euo expression. When Euo expression was reversed, the developmental cycle resumed. Additionally, this study suggests that high levels of Euo expression affect the formation of the infectious EB but not the production of the cell form committed to EB formation.

Natural reversion promotes LPS elongation in an attenuated Coxiella burnetii strain.

Lipopolysaccharide (LPS) phase variation is a critical aspect of virulence in many Gram-negative bacteria. It is of particular importance to Coxiella burnetii, the biothreat pathogen that causes Q fever, as in vitro propagation of this organism leads to LPS truncation, which is associated with an attenuated and exempted from select agent status (Nine Mile II, NMII). Here, we demonstrate that NMII was recovered from the spleens of infected guinea pigs. Moreover, these strains exhibit a previously unrecognized form of elongated LPS and display increased virulence in comparison with the initial NMII strain. The reversion of a 3-bp mutation in the gene cbu0533 directly leads to LPS elongation. To address potential safety concerns, we introduce a modified NMII strain unable to produce elongated LPS.