Biology subjects

Clemente, T. M.

Publications and source records attributed to Clemente, T. M..

3 recordsLinked to original sources

bioRxiv2023

Coxiella burnetii actively blocks IL-17-induced oxidative stress in macrophages

Coxiella burnetii is a highly infectious, aerosol-transmitted obligate intracellular bacterium that causes human Q fever and replicates within alveolar macrophages during pulmonary infection. Successful infection requires the C. burnetii Type IVB secretion system (T4BSS), which delivers bacterial effector proteins into the host cytoplasm to remodel host processes and suppress the innate immune response. In the lung, IL-17 promotes antibacterial immunity by activating ACT1-TRAF6-dependent pathways that drive inflammatory gene expression, reactive oxygen species production, and neutrophil recruitment. However, how intracellular pathogens subvert IL-17 signaling pathways to promote survival within macrophages remains poorly understood. Here, we show that C. burnetii evades IL-17-mediated host defense by targeting TRAF6, a central ubiquitin-dependent regulator of innate immune signaling. IL-17 signaling through ACT1-TRAF6 restricts C. burnetii viability in macrophages; however, C. burnetii impairs IL-17-induced NF-{kappa}B and MAPK activation, particularly JNK phosphorylation. C. burnetii T4BSS activity suppresses IL-17-driven reactive oxygen species production and neutrophil recruitment. We identify the C. burnetii T4BSS effector EmcB as a TRAF6-targeting deubiquitinase that disrupts IL-17-dependent antimicrobial and chemotactic responses. Together these findings reveal that C. burnetii downregulates intracellular innate immune signaling by targeting TRAF6, enabling evasion of the macrophage antimicrobial response and limiting neutrophil-mediated immunity. Significance StatementCoxiella burnetii causes Q fever and survives inside macrophages, immune cells that normally help eliminate inhaled pathogens. This study identifies a mechanism by which C. burnetii weakens macrophage defenses. We show that the bacterium disrupts macrophage IL-17 signaling, an immune pathway that promotes inflammatory gene expression, reactive oxygen species production, and neutrophil recruitment. Mechanistically, C. burnetii uses the secreted enzyme EmcB to target TRAF6, a central signaling protein required for IL-17-dependent antimicrobial responses. These findings reveal how an intracellular bacterial pathogen disables a key immune signaling hub to promote survival and limit innate immune activation, providing broader insight into how pathogens manipulate host immunity.

microbiology↗

bioRxiv2019

Coxiella burnetii Type 4B Secretion System-dependent manipulation of endolysosomal maturation is required for bacterial growth

Upon host cell infection, the obligate intracellular bacterium C. burnetii resides and multiplies within the Coxiella-Containing Vacuole (CCV). The nascent CCV progresses through the endosomal maturation pathway into a phagolysosome, acquiring lysosomal markers as well as acidic pH and active proteases and hydrolases. Approximately 24-48 hours post infection, heterotypic fusion between the CCV and host endosomes/lysosomes leads to CCV expansion and subsequent bacterial replication in the mature CCV. Initial CCV acidification is required to activate C. burnetii metabolism and the Type 4B Secretion System (T4BSS), which secretes effector proteins required for CCV maturation. However, we recently found that the mature CCV is less acidic (pH~5.2) than lysosomes (pH~4.8). Further, CCV acidification to pH~4.8 causes C. burnetii lysis, suggesting C. burnetii actively regulates CCV pH. Because heterotypic fusion with host endosomes/lysosomes may influence CCV pH, we investigated endosomal maturation in cells infected with wildtype (WT) or T4BSS mutant ({Delta}dotA) C. burnetii. We observed significantly fewer LAMP1-positive lysosomes, along with less acidic \"mature\" endosomes (pH~5.8), in WT-infected cells, compared to mock or {Delta}dotA-infected cells. Further, while endosomes progressively acidified from the periphery (pH~5.5) to the perinuclear area (pH~4.7) in both mock and {Delta}dotA-infected cells, endosomes did not acidify beyond pH~5.2 in WT-infected cells, indicating that the C. burnetii T4BSS inhibits endosomal maturation. Finally, increasing the number of acidic lysosomes by overexpressing the transcription factor EB inhibited C. burnetii growth, indicating lysosomes are detrimental to C. burnetii. Overall, our data suggest that C. burnetii regulates CCV pH, possibly by reducing the number of host lysosomes available for heterotypic fusion.\n\nAuthor summaryThe obligate intracellular bacterium Coxiella burnetii causes human Q fever, which manifests as a flu-like illness but can develop into a life-threatening and difficult to treat endocarditis. C. burnetii, in contrast to many other intracellular bacteria, thrives within a lysosome-like vacuole in host cells. However, we previously found that the C. burnetii vacuole is not as acidic as lysosomes and increased acidification kills the bacteria, suggesting that C. burnetii regulates the pH of its vacuole. Here, we discovered that C. burnetii blocks endosomal maturation and acidification during host cell infection, resulting in fewer lysosomes in the host cell. Moreover, increasing lysosomes in the host cells blocked C. burnetii growth. Together, our study suggests that C. burnetii regulates vacuole acidity and blocks endosomal acidification in order to produce a permissive intracellular niche.

microbiology↗

bioRxiv2018

Coxiella burnetii blocks intracellular IL-17 signaling in macrophages

Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever. Successful host cell infection requires the Coxiella Type IVB Secretion System (T4BSS), which translocates bacterial effector proteins across the vacuole membrane into the host cytoplasm, where they manipulate a variety of cell processes. To identify host cell targets of Coxiella T4BSS effector proteins, we determined the transcriptome of murine alveolar macrophages infected with a Coxiella T4BSS effector mutant. We identified a set of inflammatory genes that are significantly upregulated in T4BSS mutant-infected cells compared to mock-infected cells or cells infected with wild type (WT) bacteria, suggesting Coxiella T4BSS effector proteins downregulate expression of these genes. In addition, the IL-17 signaling pathway was identified as one of the top pathways affected by the bacteria. While previous studies demonstrated that IL-17 plays a protective role against several pathogens, the role of IL-17 during Coxiella infection is unknown. We found that IL-17 kills intracellular Coxiella in a dose-dependent manner, with the T4BSS mutant exhibiting significantly more sensitivity to IL-17 than WT bacteria. In addition, quantitative PCR confirmed increased expression of IL-17 downstream signaling genes in T4BSS mutant-infected cells compared to WT or mock-infected cells, including the pro-inflammatory cytokines I11a, Il1b and Tnfa, the chemokines Cxcl2 and Ccl5, and the antimicrobial protein Lcn2. We further confirmed that the Coxiella T4BSS downregulates macrophage CXCL2/MIP-2 and CCL5/RANTES protein levels following IL-17 stimulation. Together, these data suggest that Coxiella downregulates IL-17 signaling in a T4BSS-dependent manner in order to escape the macrophage immune response.

microbiology↗