Reactive oxygen species (ROS) are essential components of the innate immune response against intracellular bacteria and it is thought that professional phagocytes generate ROS primarily via the phagosomal NADPH oxidase machinery. However, recent studies have suggested that mitochondrial ROS (mROS) also contribute to mouse macrophage bactericidal activity, although the mechanisms linking innate immune signalling to mitochondria for mROS generation remain unclear. Here we demonstrate that engagement of a subset of Toll-like receptors (TLR1, TLR2 and TLR4) results in the recruitment of mitochondria to macrophage phagosomes and augments mROS production. This response involves translocation of a TLR signalling adaptor, tumour necrosis factor receptor-associated factor 6 (TRAF6), to mitochondria, where it engages the protein ECSIT (evolutionarily conserved signalling intermediate in Toll pathways), which is implicated in mitochondrial respiratory chain assembly. Interaction with TRAF6 leads to ECSIT ubiquitination and enrichment at the mitochondrial periphery, resulting in increased mitochondrial and cellular ROS generation. ECSIT- and TRAF6-depleted macrophages have decreased levels of TLR-induced ROS and are significantly impaired in their ability to kill intracellular bacteria. Additionally, reducing macrophage mROS levels by expressing catalase in mitochondria results in defective bacterial killing, confirming the role of mROS in bactericidal activity. These results reveal a novel pathway linking innate immune signalling to mitochondria, implicate mROS as an important component of antibacterial responses and further establish mitochondria as hubs for innate immune signalling.

/pdf/tlr-signalling-augments-macrophage-bactericidal-activity-1ehsdj8b89.pdf

TLR signalling augments macrophage bactericidal activity through mitochondrial ROS

Salmonella typhimurium is a facultative intracellular pathogen capable of surviving within phagocytic cells of the reticuloendothelial system. To identify the genes important for intracellular survival, 9516 independent Tn10 insertional mutations were isolated in a virulent strain of S. typhimurium. By using an in vitro assay for survival within macrophages, 83 Tn10 mutants have been identified that have a diminished capacity for intracellular survival (designated MS or macrophage survival mutants). All of the MS mutants are less virulent than the parent strain in vivo, demonstrating that, for Salmonella, survival within the macrophage is essential for virulence. Thirty-seven of the MS mutants have been characterized as to their phenotype, including several mutations that confer sensitivity to specific microbiocidal mechanisms of the macrophage.

https://www.pnas.org/content/pnas/83/14/5189.full.pdf

Mutants of Salmonella typhimurium that cannot survive within the macrophage are avirulent

Tuberculosis (TB), one of the oldest known human diseases. is still is one of the major causes of mortality, since two million people die each year from this malady. TB has many manifestations, affecting bone, the central nervous system, and many other organ systems, but it is primarily a pulmonary disease that is initiated by the deposition of Mycobacterium tuberculosis, contained in aerosol droplets, onto lung alveolar surfaces. From this point, the progression of the disease can have several outcomes, determined largely by the response of the host immune system. The efficacy of this response is affected by intrinsic factors such as the genetics of the immune system as well as extrinsic factors, e.g., insults to the immune system and the nutritional and physiological state of the host. In addition, the pathogen may play a role in disease progression since some M. tuberculosis strains are reportedly more virulent than others, as defined by increased transmissibility as well as being associated with higher morbidity and mortality in infected individuals. Despite the widespread use of an attenuated live vaccine and several antibiotics, there is more TB than ever before, requiring new vaccines and drugs and more specific and rapid diagnostics. Researchers are utilizing information obtained from the complete sequence of the M. tuberculosis genome and from new genetic and physiological methods to identify targets in M. tuberculosis that will aid in the development of these sorely needed antitubercular agents.

Mycobacterium tuberculosis Pathogenesis and Molecular Determinants of Virulence

Most mucosal surfaces of the mammalian body are colonized by microbial communities ("microbiota"). A high density of commensal microbiota inhabits the intestine and shields from infection ("colonization resistance"). The virulence strategies allowing enteropathogenic bacteria to successfully compete with the microbiota and overcome colonization resistance are poorly understood. Here, we investigated manipulation of the intestinal microbiota by the enteropathogenic bacterium Salmonella enterica subspecies 1 serovar Typhimurium (S. Tm) in a mouse colitis model: we found that inflammatory host responses induced by S. Tm changed microbiota composition and suppressed its growth. In contrast to wild-type S. Tm, an avirulent invGsseD mutant failing to trigger colitis was outcompeted by the microbiota. This competitive defect was reverted if inflammation was provided concomitantly by mixed infection with wild-type S. Tm or in mice (IL10(-/-), VILLIN-HA(CL4-CD8)) with inflammatory bowel disease. Thus, inflammation is necessary and sufficient for overcoming colonization resistance. This reveals a new concept in infectious disease: in contrast to current thinking, inflammation is not always detrimental for the pathogen. Triggering the host's immune defence can shift the balance between the protective microbiota and the pathogen in favour of the pathogen.

/pdf/salmonella-enterica-serovar-typhimurium-exploits-210w44stpz.pdf

Salmonella enterica serovar typhimurium exploits inflammation to compete with the intestinal microbiota

The type III secretion system of Salmonella typhimurium directs the translocation of proteins into host cells. Evolutionarily related to the flagellar assembly machinery, this system is also present in other pathogenic bacteria, but its organization is unknown. Electron microscopy revealed supramolecular structures spanning the inner and outer membranes of flagellated and nonflagellated strains; such structures were not detected in strains carrying null mutations in components of the type III apparatus. Isolated structures were found to contain at least three proteins of this secretion system. Thus, the type III apparatus of S. typhimurium, and presumably other bacteria, exists as a supramolecular structure in the bacterial envelope.

https://science.sciencemag.org/content/sci/280/5363/602.full.pdf

Supramolecular structure of the Salmonella typhimurium type III protein secretion system.

An auxotrophic mutant which requires a metabolite not available in vertebrate tissues should be unable to grow in such tissue and thus be non-virulent. Most mutations to auxotrophy do not affect virulence1–3, presumably because the required metabolites are available at sufficient concentration. However, Bacon et al.1,2 noted that a Salmonella typhi mutant requiring p-amino-benzoic acid (p AB) was less virulent (for mice) than its parent. Salmonella, unlike vertebrates, cannot assimilate exogenous folate and must synthesize it from p AB; the virtual absence of p AB from vertebrate tissues is shown by the efficacy of sulphonamide chemotherapy. Yancey et al.4 reported reduced virulence for a S. typhimurium mutant with a requirement for 2,3-dihydroxybenzoate (DHB), the precursor of the bacterial iron-acquisition compound, enterochelin. As DHB is not a known vertebrate metabolite, it would be expected to be absent from mouse tissues. Salmonella synthesize p AB and DHB from chorismate, the final product of the aromatic biosynthetic (aro) pathway. A complete block at any step of this pathway should make S. typhimurium auxotrophic for two compounds not available in vertebrate tissues, and thus non-virulent. We describe here the use of a tetracycline-resistance transposon, Tn10 (refs 5, 6), inserted in gene aroA to produce non-reverting, aromatic-requiring derivatives of virulent S. typhimurium strains. These derivatives were virtually non-virulent; their use as live vaccines conferred excellent protection against challenge with a virulent strain.

Susan K. Hoiseth

Papers

Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines.