Inducing Pyroptosis with FlaTox, RodTox, or NeedleTox.

Targeted activation of the NAIP-NLRC4 inflammasome has proven very useful in the study of pyroptosis. FlaTox and derivative LFn-NAIP-ligand cytosolic delivery systems offer a unique opportunity to interrogate both ligand recognition and downstream effects of the NAIP-NLRC4 inflammasome pathway. Here we describe how to stimulate the NAIP-NLRC4 inflammasome in vitro and in vivo. We describe experimental setup and specific considerations for treatment of macrophages in vitro and in vivo injections using a murine model of systemic inflammasome activation. The in vitro readouts of inflammasome activation propidium iodide uptake and lactate dehydrogenase (LDH) release as well as the in vivo readouts of hematocrit and body temperature measurement are described.

Human NAIP/NLRC4 and NLRP3 inflammasomes detect Salmonella type III secretion system activities to restrict intracellular bacterial replication.

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that uses two distinct type III secretion systems (T3SSs), termed Salmonella pathogenicity island (SPI)-1 and SPI-2, to deliver virulence factors into the host cell. The SPI-1 T3SS enables Salmonella to invade host cells, while the SPI-2 T3SS facilitates Salmonella's intracellular survival. In mice, a family of cytosolic immune sensors, including NAIP1, NAIP2, and NAIP5/6, recognizes the SPI-1 T3SS needle, inner rod, and flagellin proteins, respectively. Ligand recognition triggers assembly of the NAIP/NLRC4 inflammasome, which mediates caspase-1 activation, IL-1 family cytokine secretion, and pyroptosis of infected cells. In contrast to mice, humans encode a single NAIP that broadly recognizes all three ligands. The role of NAIP/NLRC4 or other inflammasomes during Salmonella infection of human macrophages is unclear. We find that although the NAIP/NLRC4 inflammasome is essential for detecting T3SS ligands in human macrophages, it is partially required for responses to infection, as Salmonella also activated the NLRP3 and CASP4/5 inflammasomes. Importantly, we demonstrate that combinatorial NAIP/NLRC4 and NLRP3 inflammasome activation restricts Salmonella replication in human macrophages. In contrast to SPI-1, the SPI-2 T3SS inner rod is not sensed by human or murine NAIPs, which is thought to allow Salmonella to evade host recognition and replicate intracellularly. Intriguingly, we find that human NAIP detects the SPI-2 T3SS needle protein. Critically, in the absence of both flagellin and the SPI-1 T3SS, the NAIP/NLRC4 inflammasome still controlled intracellular Salmonella burden. These findings reveal that recognition of Salmonella SPI-1 and SPI-2 T3SSs and engagement of both the NAIP/NLRC4 and NLRP3 inflammasomes control Salmonella infection in human macrophages.

Myoviridae phage PDX kills enteroaggregative Escherichia coli without human microbiome dysbiosis.

Introduction. Bacteriophage therapy can be developed to target emerging diarrhoeal pathogens, but doing so in the absence of microbiome disruption, which occurs with antibiotic treatment, has not been established.Aim. Identify a therapeutic bacteriophage that kills diarrhoeagenic enteroaggregative Escherichia coli (EAEC) while leaving the human microbiome intact.Methodology. Phages from wastewater in Portland, OR, USA were screened for bacteriolytic activity by overlay assay. One isolated phage, PDX, was classified by electron microscopy and genome sequencing. A mouse model of infection determined whether the phage was therapeutic against EAEC. 16S metagenomic analysis of anaerobic cultures determined whether a normal human microbiome was altered by treatment.Results. Escherichia virus PDX, a member of the strictly lytic family Myoviridae, killed a case-associated EAEC isolate from a child in rural Tennessee in a dose-dependent manner, and killed EAEC isolates from Columbian children. A single dose of PDX (multiplicity of infection: 100) 1 day post-infection reduced EAEC recovered from mouse faeces. PDX also killed EAEC when cultured anaerobically in the presence of human faecal bacteria. While the addition of EAEC reduced the ß-diversity of the human microbiota, that of the cultures with either faeces alone, faeces with EAEC and PDX, or with just PDX phage was not different statistically.Conclusion. PDX killed EAEC isolate EN1E-0007 in vivo and in vitro, while not altering the diversity of normal human microbiota in anaerobic culture, and thus could be part of an effective therapy for children in developing countries and those suffering from EAEC-mediated traveller's diarrhoea without causing dysbiosis.

Effect of ULV malathion use in boll weevil (Coleoptera: Curculionidae) eradication on resistance in the tarnished plant bug (Heteroptera: Miridae).

Tarnished plant bugs, Lygus lineolaris (Palisot de Beauvois), from regions 1, 2, and 3 of the boll weevil, Anthonomous grandis Boheman, eradication program in Mississippi were collected from wild hosts and tested for malathion resistance during the spring and fall of 2000 and 2001. Plant bugs were also tested in region 1 in late-July and October of 1999, just before and after multiple applications of ultra-low-volume (ULV) malathion were used for reproduction-diapause control of boll weevils in August and September. Regions 1 (north Delta), 2 (south Delta), and 3 (hills) began boll weevil eradication in 1999, 1998, and 1997, respectively. A glass-vial bioassay was used to determine resistance in plant bugs to malathion by comparing LC50 values against an LC50 value obtained for susceptible plant bugs. Comparison of the LC50 value obtained for plant bugs at a location in the spring was also made with the LC50 value obtained in the fall at the same location. After multiple applications of malathion made for reproduction-diapause boll weevil control in region 1 in August and September, malathion resistance increased by 4.9-, 6.5-, and 20.8-fold in plant bug populations from the three test locations. Results from testing bugs from all three eradication regions were similar. Malathion resistance usually increased significantly from spring to fall and then declined significantly from fall to spring of the next year. Despite reduced use of malathion in all three eradication regions for boll weevils in 2001, resistance to malathion in plant bugs still increased significantly from spring to fall at all test locations in regions 1 and 2 (the Delta). Malathion resistance did not increase significantly in plant bug populations in region 3 (the hills) in 2001 from spring to fall at three of four test locations in this year. Possible causes for the higher malathion resistance found in plant bugs in the Delta are discussed. Overall test results showed that the use of malathion in boll weevil eradication in cotton probably contributed to increases in resistance to malathion in plant bug populations in the eradication areas. However, the expression of this resistance was usually rapidly lost by spring of the following year. Boll weevil eradication did not seem to produce a permanent increase in the expression of malathion resistance in tarnished plant bug populations found in the eradication regions.