Summary
Activation of the DNA-dependent cytosolic surveillance pathway in response to Mycobacterium tuberculosis infection stimulates ubiquitin-dependent autophagy and inflammatory cytokine production, and plays an important role in host defense against M. tuberculosis. However, the identity of the host sensor for M. tuberculosis DNA is unknown. Here we show that M. tuberculosis activated cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) synthase (cGAS) in macrophages to produce cGAMP, a second messenger that activates the adaptor protein stimulator of interferon genes (STING) to induce type I interferons and other cytokines. cGAS localized with M. tuberculosis in mouse and human cells and in human tuberculosis lesions. Knockdown or knockout of cGAS in human or mouse macrophages blocked cytokine production and induction of autophagy. Mice deficient in cGAS were more susceptible to lethality caused by infection with M. tuberculosis. These results demonstrate that cGAS is a vital innate immune sensor of M. tuberculosis infection.
SUMMARY
During antibacterial autophagy, ubiquitination of intracellular bacteria recruits proteins that mediate bacterial delivery to the lysosome for degradation. Smurf1 is an E3 ubiquitin-ligase whose role in selective bacterial autophagy is unknown. We show that Smurf1 facilitates selective autophagy of the human pathogen Mycobacterium tuberculosis (Mtb). Smurf1−/− macrophages are defective in recruiting polyubiquitin, the proteasome, the ubiquitin-binding autophagy adaptor NBR1, the autophagy protein LC3, and the lysosomal marker LAMP1 to Mtb-associated structures, and are more permissive for Mtb growth. This function of Smurf1 requires both its ubiquitin-ligase and C2 phospholipid-binding domains, and involves K48-rather than K63-linked ubiquitination. Chronically infected Smurf1−/− mice have increased bacterial load, increased lung inflammation, and accelerated mortality. SMURF1 controls Mtb replication in human macrophages and associates with bacteria in lungs of patients with pulmonary tuberculosis. Thus, Smurf1 is required for selective autophagy of Mtb and host defense against tuberculosis infection.
Mycobacterium tuberculosis (Mtb), the causative agent of
tuberculosis, is responsible for 1.5 million deaths annually. We previously
showed that Mtb infection in mice induces expression of the carbon monoxide (CO)
producing enzyme heme oxygenase (HO1) and that CO is sensed by Mtb to initiate a
dormancy program. Further, mice deficient in HO1 succumb to Mtb infection more
readily than wild type mice. While mouse macrophages control intracellular Mtb
infection through several mechanisms such as nitric oxide synthase, the
respiratory burst, acidification and autophagy, how human macrophages control
Mtb infection remains less well understood. Here we show that Mtb induces and
colocalizes with HO1 in both mouse and human tuberculosis lesions in
vivo, and that Mtb induces and colocalizes with HO1 during primary
human macrophage infection in vitro. Surprisingly, we find that
chemical inhibition of HO1 both reduces inflammatory cytokine production by
human macrophages and restricts intracellular growth of mycobacteria. Thus,
induction of HO1 by Mtb infection may be a mycobacterial virulence mechanism to
enhance inflammation and bacterial growth.
A reader brought errors in Figure 4 to the authors' attention. In Figures 4A and 4D, several immunofluorescence panels were inadvertently reversed during their final arrangement. Panels that should have indicated the merge image were incorrectly placed where the single channel GFP image should be, and vice versa. Figures 4A and 4D have now been corrected online. The original results and conclusions are unaffected by these corrections. The authors accept responsibility for not detecting these errors prior to publication and regret any inconvenience this has caused.
bMethicillin-resistant Staphylococcus aureus (MRSA) is a well-known public health concern. However, the means by which methicillin resistance genes are transferred among staphylococci in nature remains unknown. Older scientific literature suggests transduction as a means of mecA transfer, but the optimal conditions are reported to require plasmids and potentially a lysogenic phage. These reports preceded discovery of the staphylococcal cassette chromosome mec (SCCmec) elements. We undertook studies to confirm and clarify the conditions promoting transduction of SCCmec in S. aureus populations using well-characterized donor and recipient strains primarily of the USA300 lineage. Both bacteriophages 80␣ and 29 were capable of transducing SCCmec type IV and SCCmec type I to recipient strains of S. aureus. Pulsed-field gel electrophoresis and mec-associated dru typing were used to confirm the identity of the transductants. Transfer of mecA via transduction occurred at low frequency and required extended selection times for mecA gene expression and the presence of a penicillinase plasmid in the recipient. However, interference with the process by clavulanic acid and the necessity of lysogeny with 11 in the recipient or the presence of a small (4-kb) tetracycline resistance plasmid, as previously reported, were not confirmed. SCCmec transduction was occasionally associated with substantial deletions or truncation of SCCmec and the arginine catabolic metabolic element in USA300 recipients. Overall, these data clarify the conditions required for SCCmec transduction and document that rearrangements may occur during the process.
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