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.

Broad detection of bacterial type III secretion system and flagellin proteins by the human NAIP/NLRC4 inflammasome.

Inflammasomes are cytosolic multiprotein complexes that initiate host defense against bacterial pathogens by activating caspase-1-dependent cytokine secretion and cell death. In mice, specific nucleotide-binding domain, leucine-rich repeat-containing family, apoptosis inhibitory proteins (NAIPs) activate the nucleotide-binding domain, leucine-rich repeat-containing family, CARD domain-containing protein 4 (NLRC4) inflammasome upon sensing components of the type III secretion system (T3SS) and flagellar apparatus. NAIP1 recognizes the T3SS needle protein, NAIP2 recognizes the T3SS inner rod protein, and NAIP5 and NAIP6 recognize flagellin. In contrast, humans encode a single functional NAIP, raising the question of whether human NAIP senses one or multiple bacterial ligands. Previous studies found that human NAIP detects both flagellin and the T3SS needle protein and suggested that the ability to detect both ligands was achieved by multiple isoforms encoded by the single human NAIP gene. Here, we show that human NAIP also senses the Salmonella Typhimurium T3SS inner rod protein PrgJ and that T3SS inner rod proteins from multiple bacterial species are also detected. Furthermore, we show that a single human NAIP isoform is capable of sensing the T3SS inner rod, needle, and flagellin. Our findings indicate that, in contrast to murine NAIPs, promiscuous recognition of multiple bacterial ligands is conferred by a single human NAIP.

Restriction of arginine induces antibiotic tolerance in Staphylococcus aureus.

Staphylococcus aureus is responsible for a substantial number of invasive infections globally each year. These infections are problematic because they are frequently recalcitrant to antibiotic treatment. Antibiotic tolerance, the ability of bacteria to persist despite normally lethal doses of antibiotics, contributes to antibiotic treatment failure in S. aureus infections. To understand how antibiotic tolerance is induced, S. aureus biofilms exposed to multiple anti-staphylococcal antibiotics are examined using both quantitative proteomics and transposon sequencing. These screens indicate that arginine metabolism is involved in antibiotic tolerance within a biofilm and support the hypothesis that depletion of arginine within S. aureus communities can induce antibiotic tolerance. Consistent with this hypothesis, inactivation of argH, the final gene in the arginine synthesis pathway, induces antibiotic tolerance. Arginine restriction induces antibiotic tolerance via inhibition of protein synthesis. In murine skin and bone infection models, an argH mutant has enhanced ability to survive antibiotic treatment with vancomycin, highlighting the relationship between arginine metabolism and antibiotic tolerance during S. aureus infection. Uncovering this link between arginine metabolism and antibiotic tolerance has the potential to open new therapeutic avenues targeting previously recalcitrant S. aureus infections.

Coordinated adaptation of Staphylococcus aureus to calprotectin-dependent metal sequestration.

The host protein calprotectin inhibits the growth of a variety of bacterial pathogens through metal sequestration in a process known as "nutritional immunity." Staphylococcus aureus growth is inhibited by calprotectin in vitro, and calprotectin is localized in vivo to staphylococcal abscesses during infection. However, the staphylococcal adaptations that provide defense against nutritional immunity and the role of metal-responsive regulators are not fully characterized. In this work, we define the transcriptional response of S. aureus and the role of the metal-responsive regulators, Zur, Fur, and MntR, in response to metal limitation by calprotectin exposure. Additionally, we identified genes affecting the fitness of S. aureus during metal limitation through a Transposon sequencing (Tn-seq) approach. Loss of function mutations in clpP, which encodes a proteolytic subunit of the ATP-dependent Clp protease, demonstrate reduced fitness of S. aureus to the presence of calprotectin. ClpP contributes to pathogenesis in vivo in a calprotectin-dependent manner. These studies establish a critical role for ClpP to combat metal limitation by calprotectin and reveal the genes required for S. aureus to outcompete the host for metals. IMPORTANCE: Staphylococcus aureus is a leading cause of skin and soft tissue infections, bloodstream infections, and endocarditis. Antibiotic treatment failures during S. aureus infections are increasingly prevalent, highlighting the need for novel antimicrobial agents. Metal chelator-based therapeutics have tremendous potential as antimicrobials due to the strict requirement for nutrient metals exhibited by bacterial pathogens. The high-affinity transition metal-binding properties of calprotectin represents a potential therapeutic strategy that functions through metal chelation. Our studies provide a foundation to define mechanisms by which S. aureus combats nutritional immunity and may be useful for the development of novel therapeutics to counter the ability of S. aureus to survive in a metal-limited environment.

Listeria monocytogenes requires cellular respiration for NAD+ regeneration and pathogenesis.

Cellular respiration is essential for multiple bacterial pathogens and a validated antibiotic target. In addition to driving oxidative phosphorylation, bacterial respiration has a variety of ancillary functions that obscure its contribution to pathogenesis. We find here that the intracellular pathogen Listeria monocytogenes encodes two respiratory pathways which are partially functionally redundant and indispensable for pathogenesis. Loss of respiration decreased NAD+ regeneration, but this could be specifically reversed by heterologous expression of a water-forming NADH oxidase (NOX). NOX expression fully rescued intracellular growth defects and increased L. monocytogenes loads >1000-fold in a mouse infection model. Consistent with NAD+ regeneration maintaining L. monocytogenes viability and enabling immune evasion, a respiration-deficient strain exhibited elevated bacteriolysis within the host cytosol and NOX expression rescued this phenotype. These studies show that NAD+ regeneration represents a major role of L. monocytogenes respiration and highlight the nuanced relationship between bacterial metabolism, physiology, and pathogenesis.

Cellular respiration is one of the main ways organisms make energy. It works by linking the oxidation of an electron donor (like sugar) to the reduction of an electron acceptor (like oxygen). Electrons pass between the two molecules along what is known as an 'electron transport chain'. This process generates a force that powers the production of adenosine triphosphate (ATP), a molecule that cells use to store energy. Respiration is a common way for cells to replenish their energy stores, but it is not the only way. A simpler process that does not require a separate electron acceptor or an electron transport chain is called fermentation. Many bacteria have the capacity to perform both respiration and fermentation and do so in a context-dependent manner. Research has shown that respiration can contribute to bacterial diseases, like tuberculosis and listeriosis (a disease caused by the foodborne pathogen Listeria monocytogenes). Indeed, some antibiotics even target bacterial respiration. Despite being often discussed in the context of generating ATP, respiration is also important for many other cellular processes, including maintaining the balance of reduced and oxidized nicotinamide adenine dinucleotide (NAD) cofactors. Because of these multiple functions, the exact role respiration plays in disease is unknown. To find out more, Rivera-Lugo, Deng et al. developed strains of the bacterial pathogen Listeria monocytogenes that lacked some of the genes used in respiration. The resulting bacteria were still able to produce energy, but they became much worse at infecting mammalian cells. The use of a genetic tool that restored the balance of reduced and oxidized NAD cofactors revived the ability of respiration-deficient L. monocytogenes to infect mammalian cells, indicating that this balance is what the bacterium requires to infect. Research into respiration tends to focus on its role in generating ATP. But these results show that for some bacteria, this might not be the most important part of the process. Understanding the other roles of respiration could change the way that researchers develop antibacterial drugs in the future. This in turn could help with the growing problem of antibiotic resistance.

Stimulation of a protease targeting the LRIM1/APL1C complex reveals specificity in complement-like pathway activation in Anopheles gambiae.

The complement-like pathway of the African malaria mosquito Anopheles gambiae provides protection against infection by diverse pathogens. A functional requirement for a core set of proteins during infections by rodent and human malaria parasites, bacteria, and fungi suggests a similar mechanism operates against different pathogens. However, the extent to which the molecular mechanisms are conserved is unknown. In this study we probed the biochemical responses of complement-like pathway to challenge by the Gram-positive bacterium Staphyloccocus aureus. Western blot analysis of the hemolymph revealed that S. aureus challenge activates a TEP1 convertase-like activity and promotes the depletion of the protein SPCLIP1. S. aureus challenge did not lead to an apparent change in the abundance of the LRIM1/APL1C complex compared to challenge by the Gram-negative bacterium, Escherichia coli. Following up on this observation using a panel of LRIM1 and APL1C antibodies, we found that E. coli challenge, but not S. aureus, specifically activates a protease that cleaves the C-terminus of APL1C. Inhibitor studies in vivo and in vitro protease assays suggest that a serine protease is responsible for APL1C cleavage. This study reveals that despite different challenges converging on activation of a TEP1 convertase-like activity, the mosquito complement-like pathway also includes pathogen-specific reactions.