Summary Ubiquitin-mediated targeting of intracellular bacteria to the autophagy pathway is a key innate defense mechanism against invading microbes, including the important human pathogen Mycobacterium tuberculosis. However, the ubiquitin ligases responsible for catalyzing ubiquitin chains that surround intracellular bacteria are poorly understood. PARKIN is a ubiquitin ligase with a well-established role in mitophagy, and mutations in the PARKIN gene (Park2) lead to increased susceptibility to Parkinson’s disease. Surprisingly, genetic polymorphisms in the Park2 regulatory region are also associated with increased susceptibility to intracellular bacterial pathogens in humans, including Mycobacterium leprae and Salmonella typhi, but the function of PARKIN in immunity remains unexplored. Here we show that PARKIN plays a role in ubiquitin-mediated autophagy of M. tuberculosis. Both PARKIN-deficient mice and flies are sensitive to various intracellular bacterial infections, suggesting PARKIN plays a conserved role in metazoan innate defense. Moreover, our work reveals an unexpected functional link between mitophagy and infectious disease.

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 Mycobacterium tuberculosis (Mtb), the primary causative agent of human tuberculosis, has killed more people than any other bacterial pathogen in human history and remains one of the most important transmissible diseases worldwide. Because of the longstanding interaction of Mtb with humans, it is no surprise that human mucosal and innate immune cells have evolved multiple mechanisms to detect Mtb during initial contact. To that end, the cell surface of human cells is decorated with numerous pattern recognition receptors for a variety of mycobacterial ligands. Further, once Mtb is ingested into professional phagocytes, other host molecules are engaged to report on the presence of an intracellular pathogen. In this review, we discuss the role of specific mycobacterial products in modulating the host’s ability to detect Mtb. In addition, we describe the specific host receptors that mediate the detection of mycobacterial infection and the role of individual receptors in mycobacterial pathogenesis in humans and model organisms.

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.

BackgroundChildren with acute hematogenous osteomyelitis (AHO) have a broad spectrum of illness ranging from mild to severe. The purpose of this study is to evaluate the impact of genomic variation of Staphylococcus aureus on clinical phenotype of affected children and determine which virulence genes correlate with severity of illness.MethodsDe novo whole genome sequencing was conducted for a strain of Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA), using PacBio Hierarchical Genome Assembly Process (HGAP) from 6 Single Molecule Real Time (SMRT) Cells, as a reference for DNA library assembly of 71 Staphylococcus aureus isolates from children with AHO. Virulence gene annotation was based on exhaustive literature review and genomic data in NCBI for Staphylococcus aureus. Clinical phenotype was assessed using a validated severity score. Kruskal-Wallis rank sum test determined association between clinical severity and virulence gene presence using False Discovery Rate (FDR), significance <0.01.ResultsPacBio produced an assembled genome of 2,898,306 bp and 2054 Open Reading Frames (ORFs). Annotation confirmed 201 virulence genes. Statistical analysis of gene presence by clinical severity found 40 genes significantly associated with severity of illness (FDR ≤0.009). MRSA isolates encoded a significantly greater number of virulence genes than did MSSA (p < 0.0001). Phylogenetic analysis by maximum likelihood (PAML) demonstrated the relatedness of genomic distance to clinical phenotype.ConclusionsThe Staphylococcus aureus genome contains virulence genes which are significantly associated with severity of illness in children with osteomyelitis. This study introduces a novel reference strain and detailed annotation of Staphylococcus aureus virulence genes. While this study does not address bacterial gene expression, a platform is created for future transcriptome investigations to elucidate the complex mechanisms involved in childhood osteomyelitis.

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Mycobacterium tuberculosis, the causative agent of tuberculosis, is a major cause of morbidity and mortality worldwide. However, an effective vaccine for M. tuberculosis is lacking. We panned a phage display library using monoclonal antibodies against M. tuberculosis liporabinomannan (LAM), an important component of the M. tuberculosis cell wall, and identified two peptide sequences, HSFKWLDSPRLR or SGVYKVAYDWQH, with high antibody affinity after multiple rounds of panning. Only the HSFKWLDSPRLR peptide induced an anti-LAM response when conjugated to either keyhole limpet hemocyanin (KLH) or to the baculovirus Autographa californica multicapsid nucleopolyherovirus (AcMNPV) when introduced into mice by injection or via intranasal inoculation, respectively. Vaccination with AcMNPV conjugated HSFKWLDSPRLR peptide delayed mortality in a mouse model of tuberculosis. Thus, we report a proof of principle M. tuberculosis vaccination strategy combining an anti-LAM mimotope with a baculovirus delivery system.

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