Differential survival of Staphylococcal species in macrophages.

Staphylococcus aureus is considered an extracellular pathogen, yet the bacterium is able to survive within and escape from host cells. An agr/sae mutant of strain USA300 is unable to escape from macrophages but can replicate and survive within. We questioned whether such "non-toxic" S. aureus resembles the less pathogenic coagulase-negative Staphylococcal (CoNS) species like S. epidermidis, S. carnosus, S. lugdunensis, S. capitis, S. warneri, or S. pettenkoferi. We show that the CoNS are more efficiently killed in macrophage-like THP-1 cells or in human primary macrophages. Mutations in katA, copL, the regulatory system graRS, or sigB did not impact bacterial survival in THP-1 cells. Deletion of the superoxide dismutases impaired S. aureus survival in primary macrophages but not in THP-1 cells. However, expression of the S. aureus-specific sodM in S. epidermidis was not sufficient to protect this species from being killed. Thus, at least in those cells, better bacterial survival of S. aureus could not be linked to higher protection from ROS. However, "non-toxic" S. aureus was found to be insensitive to pH, whereas most CoNS were protected when phagosomal acidification was inhibited. Thus, species differences are at least partially linked to differences in sensitivity to acidification.

Human NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome activity is regulated by and potentially targetable through Bruton tyrosine kinase.

BACKGROUND: The Nod-like receptor NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK) are protagonists in innate and adaptive immunity, respectively. NLRP3 senses exogenous and endogenous insults, leading to inflammasome activation, which occurs spontaneously in patients with Muckle-Wells syndrome; BTK mutations cause the genetic immunodeficiency X-linked agammaglobulinemia (XLA). However, to date, few proteins that regulate NLRP3 inflammasome activity in human primary immune cells have been identified, and clinically promising pharmacologic targeting strategies remain elusive. OBJECTIVE: We sought to identify novel regulators of the NLRP3 inflammasome in human cells with a view to exploring interference with inflammasome activity at the level of such regulators. METHODS: After proteome-wide phosphoproteomics, the identified novel regulator BTK was studied in human and murine cells by using pharmacologic and genetic BTK ablation. RESULTS: Here we show that BTK is a critical regulator of NLRP3 inflammasome activation: pharmacologic (using the US Food and Drug Administration-approved inhibitor ibrutinib) and genetic (in patients with XLA and Btk knockout mice) BTK ablation in primary immune cells led to reduced IL-1ß processing and secretion in response to nigericin and the Staphylococcus aureus toxin leukocidin AB (LukAB). BTK affected apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and caspase-1 cleavage and interacted with NLRP3 and ASC. S aureus infection control in vivo and IL-1ß release from cells of patients with Muckle-Wells syndrome were impaired by ibrutinib. Notably, IL-1ß processing and release from immune cells isolated from patients with cancer receiving ibrutinib therapy were reduced. CONCLUSION: Our data suggest that XLA might result in part from genetic inflammasome deficiency and that NLRP3 inflammasome-linked inflammation could potentially be targeted pharmacologically through BTK.

Influence of Sae-regulated and Agr-regulated factors on the escape of Staphylococcus aureus from human macrophages.

Although Staphylococcus aureus is not a classical intracellular pathogen, it can survive within phagocytes and many other cell types. However, the pathogen is also able to escape from cells by mechanisms that are only partially understood. We analysed a series of isogenic S. aureus mutants of the USA300 derivative JE2 for their capacity to destroy human macrophages from within. Intracellular S. aureus JE2 caused severe cell damage in human macrophages and could efficiently escape from within the cells. To obtain this full escape phenotype including an intermittent residency in the cytoplasm, the combined action of the regulatory systems Sae and Agr is required. Mutants in Sae or mutants deficient in the Sae target genes lukAB and pvl remained in high numbers within the macrophages causing reduced cell damage. Mutants in the regulatory system Agr or in the Agr target gene psmα were largely similar to wild-type bacteria concerning cell damage and escape efficiency. However, these strains were rarely detectable in the cytoplasm, emphasizing the role of phenol-soluble modulins (PSMs) for phagosomal escape. Thus, Sae-regulated toxins largely determine damage and escape from within macrophages, whereas PSMs are mainly responsible for the escape from the phagosome into the cytoplasm. Damage of macrophages induced by intracellular bacteria was linked neither to activation of apoptosis-related caspase 3, 7 or 8 nor to NLRP3-dependent inflammasome activation.