Constitutive secretion of pro-IL-18 allows keratinocytes to initiate inflammation during bacterial infection.

Group A Streptococcus (GAS, Streptococcus pyogenes) is a professional human pathogen that commonly infects the skin. Keratinocytes are one of the first cells to contact GAS, and by inducing inflammation, they can initiate the earliest immune responses to pathogen invasion. Here, we characterized the proinflammatory cytokine repertoire produced by primary human keratinocytes and surrogate cell lines commonly used in vitro. Infection induces several cytokines and chemokines, but keratinocytes constitutively secrete IL-18 in a form that is inert (pro-IL-18) and lacks proinflammatory activity. Canonically, IL-18 activation and secretion are coupled through a single proteolytic event that is regulated intracellularly by the inflammasome protease caspase-1 in myeloid cells. The pool of extracellular pro-IL-18 generated by keratinocytes is poised to sense extracellular proteases. It is directly processed into a mature active form by SpeB, a secreted GAS protease that is a critical virulent factor during skin infection. This mechanism contributes to the proinflammatory response against GAS, resulting in T cell activation and the secretion of IFN-γ. Under these conditions, isolates of several other major bacterial pathogens and microbiota of the skin were found to not have significant IL-18-maturing ability. These results suggest keratinocyte-secreted IL-18 is a sentinel that sounds an early alarm that is highly sensitive to GAS, yet tolerant to non-invasive members of the microbiota.

Assessing Interleukin-1ß Activation During Pyroptosis.

The cytokine interleukin-1ß (IL-1ß) is a major mediator of inflammation in autoinflammatory disease and the host response to infection. IL-1ß is stored within cells in an inert form, which requires proteolytic removal of an amino-terminal fragment in order to bind the IL-1 receptor complex and have pro-inflammatory activity. This cleavage event is canonically carried out by inflammasome-activated caspase proteases, but microbe and host proteases can also generate unique active forms. Post-translational regulation of IL-1ß and the diversity in resulting products can present challenges to the evaluation of IL-1ß activation. This chapter details methods and important controls for the accurate and sensitive measurement of IL-1ß activation within biological samples.

Chemical Optimization of Selective Pseudomonas aeruginosa LasB Elastase Inhibitors and Their Impact on LasB-Mediated Activation of IL-1ß in Cellular and Animal Infection Models.

LasB elastase is a broad-spectrum exoprotease and a key virulence factor of Pseudomonas aeruginosa, a major pathogen causing lung damage and inflammation in acute and chronic respiratory infections. Here, we describe the chemical optimization of specific LasB inhibitors with druglike properties and investigate their impact in cellular and animal models of P. aeruginosa infection. Competitive inhibition of LasB was demonstrated through structural and kinetic studies. In vitro LasB inhibition was confirmed with respect to several host target proteins, namely, elastin, IgG, and pro-IL-1ß. Furthermore, inhibition of LasB-mediated IL-1ß activation was demonstrated in macrophage and mouse lung infection models. In mice, intravenous administration of inhibitors also resulted in reduced bacterial numbers at 24 h. These highly potent, selective, and soluble LasB inhibitors constitute valuable tools to study the proinflammatory impact of LasB in P. aeruginosa infections and, most importantly, show clear potential for the clinical development of a novel therapy for life-threatening respiratory infections caused by this opportunistic pathogen.

Group A Streptococcus induces GSDMA-dependent pyroptosis in keratinocytes.

Gasdermins (GSDMs) are a family of pore-forming effectors that permeabilize the cell membrane during the cell death program pyroptosis1. GSDMs are activated by proteolytic removal of autoinhibitory carboxy-terminal domains, typically by caspase regulators1-9. However, no activator is known for one member of this family, GSDMA. Here we show that the major human pathogen group A Streptococcus (GAS) secretes a protease virulence factor, SpeB, that induces GSDMA-dependent pyroptosis. SpeB cleavage of GSDMA releases an active amino-terminal fragment that can insert into membranes to form lytic pores. GSDMA is primarily expressed in the skin10, and keratinocytes infected with SpeB-expressing GAS die of GSDMA-dependent pyroptosis. Mice have three homologues of human GSDMA, and triple-knockout mice are more susceptible to invasive infection by a pandemic hypervirulent M1T1 clone of GAS. These results indicate that GSDMA is critical in the immune defence against invasive skin infections by GAS. Furthermore, they show that GSDMs can act independently of host regulators as direct sensors of exogenous proteases. As SpeB is essential for tissue invasion and survival within skin cells, these results suggest that GSDMA can act akin to a guard protein that directly detects concerning virulence activities of microorganisms that present a severe infectious threat.

Restoring Endogenous Repair Mechanisms to Heal Chronic Wounds with a Multifunctional Wound Dressing.

Current treatment of chronic wounds has been critically limited by various factors, including bacterial infection, biofilm formation, impaired angiogenesis, and prolonged inflammation. Addressing these challenges, we developed a multifunctional wound dressing-based three-pronged approach for accelerating wound healing. The multifunctional wound dressing, composed of nanofibers, functional nanoparticles, natural biopolymers, and selected protein and peptide, can target multiple endogenous repair mechanisms and represents a promising alternative to current wound healing products.

The Pseudomonas aeruginosa protease LasB directly activates IL-1ß.

BACKGROUND: Pulmonary damage by Pseudomonas aeruginosa during cystic fibrosis lung infection and ventilator-associated pneumonia is mediated both by pathogen virulence factors and host inflammation. Impaired immune function due to tissue damage and inflammation, coupled with pathogen multidrug resistance, complicates the management of these deep-seated infections. Pathological inflammation during infection is driven by interleukin-1ß (IL-1ß), but the molecular processes involved are not fully understood. METHODS: We examined IL-1ß activation in a pulmonary model infection of Pseudomonas aeruginosa and in vitro using genetics, specific inhibitors, recombinant proteins, and targeted reporters of protease activity and IL-1ß bioactivity. FINDINGS: Caspase-family inflammasome proteases canonically regulate maturation of this proinflammatory cytokine, but we report that plasticity in IL-1ß proteolytic activation allows for its direct maturation by the pseudomonal protease LasB. LasB promotes IL-1ß activation, neutrophilic inflammation, and destruction of lung architecture characteristic of severe P. aeruginosa pulmonary infection. INTERPRETATION: Preservation of lung function and effective immune clearance may be enhanced by selectively controlling inflammation. Discovery of this IL-1ß regulatory mechanism provides a distinct target for anti-inflammatory therapeutics, such as matrix metalloprotease inhibitors that inhibit LasB and limit inflammation and pathology during P. aeruginosa pulmonary infections. FUNDING: Full details are provided in the Acknowledgements section.

Group A Streptococcus Infection of the Nasopharynx Requires Proinflammatory Signaling through the Interleukin-1 Receptor.

Group A Streptococcus (GAS) is the etiologic agent of numerous high-morbidity and high-mortality diseases. Infections are typically highly proinflammatory. During the invasive infection necrotizing fasciitis, this is in part due to the GAS protease SpeB directly activating interleukin-1ß (IL-1ß) independent of the canonical inflammasome pathway. The upper respiratory tract is the primary site for GAS colonization, infection, and transmission, but the host-pathogen interactions at this site are still largely unknown. We found that in the murine nasopharynx, SpeB enhanced IL-1ß-mediated inflammation and the chemotaxis of neutrophils. However, neutrophilic inflammation did not restrict infection and instead promoted GAS replication and disease. Inhibiting IL-1ß or depleting neutrophils, which both promote invasive infection, prevented GAS infection of the nasopharynx. Mice pretreated with penicillin became more susceptible to GAS challenge, and this reversed the attenuation from neutralization or depletion of IL-1ß, neutrophils, or SpeB. Collectively, our results suggest that SpeB is essential to activate an IL-1ß-driven neutrophil response. Unlike during invasive tissue infections, this is beneficial in the upper respiratory tract because it disrupts colonization resistance mediated by the microbiota. This provides experimental evidence that the notable inflammation of strep throat, which presents with significant swelling, pain, and neutrophil influx, is not an ineffectual immune response but rather is a GAS-directed remodeling of this niche for its pathogenic benefit.

Inflammasome inhibition blocks cardiac glycoside cell toxicity.

Chronic heart failure and cardiac arrhythmias have high morbidity and mortality, and drugs for the prevention and management of these diseases are a large part of the pharmaceutical market. Among these drugs are plant-derived cardiac glycosides, which have been used by various cultures over millennia as both medicines and poisons. We report that digoxin and related compounds activate the NLRP3 inflammasome in macrophages and cardiomyocytes at concentrations achievable during clinical use. Inflammasome activation initiates the maturation and release of the inflammatory cytokine IL-1ß and the programmed cell death pathway pyroptosis in a caspase-1-dependent manner. Notably, the same fluxes of potassium and calcium cations that affect heart contraction also induce inflammasome activation in human but not murine cells. Pharmaceuticals that antagonize these fluxes, including glyburide and verapamil, also inhibit inflammasome activation by cardiac glycosides. Cardiac glycoside-induced cellular cytotoxicity and IL-1ß signaling are likewise antagonized by inhibitors of the NLRP3 inflammasome or the IL-1 receptor-targeting biological agent anakinra. Our results inform on the molecular mechanism by which the inflammasome integrates the diverse signals that activate it through secondary signals like cation flux. Furthermore, this mechanism suggests a contribution of the inflammasome to the toxicity and adverse events associated with cardiac glycosides use in humans and that targeted anti-inflammatories could provide an additional adjunct therapeutic countermeasure.

The Mla pathway is critical for Pseudomonas aeruginosa resistance to outer membrane permeabilization and host innate immune clearance.

Pseudomonas aeruginosa is an important opportunistic pathogen that has become a serious problem due to increased rates of antibiotic resistance. Due to this along with a dearth in novel antibiotic development, especially against Gram-negative pathogens, new therapeutic strategies are needed to prevent a post-antibiotic era. Here, we describe the importance of the vacJ/Mla pathway in resisting bactericidal actions of the host innate immune response. P. aeruginosa tn5 transposon mutants in genes from the VacJ/Mla pathway showed increased susceptibility to killing by the host cathelicidin antimicrobial peptide, LL-37, when compared to the wild-type parent strain. The P. aeruginosa vacJ - mutant demonstrated increased membrane permeability upon damage as well as sensitivity to killing in the presence of the detergent sodium dodecyl sulfate and the divalent cation chelator EDTA. When exposed to human whole blood and serum complement, the vacJ - mutant was killed more rapidly when compared to the wild-type parent strain and complemented mutant. Finally, in an in vivo mouse lung infection model, infection with the vacJ - mutant resulted in reduced mortality, lower bacterial burden, and reduced lung damage when compared to the wild-type strain. This study highlights the potential in therapeutically targeting the VacJ/Mla pathway in sensitizing P. aeruginosa to killing by the host innate immune response. KEY MESSAGES: • The Mla pathway regulates outer membrane dynamics in human pathogen Pseudomonas aeruginosa (PA). • Disruption of Mla pathway gene vacJ sensitizes PA to host cathelicidin antimicrobial peptide LL-37. • Loss of vacJ expression renders PA more sensitive to human whole blood and serum killing. • Loss of vacJ expression reduces PA survival and virulence in a murine lung infection model. • The Mla pathway merits exploration as a pharmacologic target to sensitize PA to host innate immunity.

IL-1ß is an innate immune sensor of microbial proteolysis.

Interleukin-1ß (IL-1ß) is a key proinflammatory cytokine that drives antimicrobial immune responses. IL-1ß is aberrantly activated in autoimmune diseases, and IL-1ß inhibitors are used as therapeutic agents to treat patients with certain autoimmune disorders. Review of postmarketing surveillance of patients receiving IL-1ß inhibitors found a disproportionate reporting of invasive infections by group A Streptococcus (GAS). IL-1ß inhibition increased mouse susceptibility to GAS infection, but IL-1ß was produced independent of canonical inflammasomes. Newly synthesized IL-1ß has an amino-terminal prodomain that blocks signaling activity, which is usually proteolytically removed by caspase-1, a protease activated within the inflammasome structure. In place of host caspases, the secreted GAS cysteine protease SpeB generated mature IL-1ß. During invasive infection, GAS isolates may acquire pathoadaptive mutations eliminating SpeB expression to evade detection by IL-1ß. Pharmacological IL-1ß inhibition alleviates this selective pressure, allowing invasive infection by nonpathoadapted GAS. Thus, IL-1ß is a sensor that directly detects pathogen-associated proteolysis through an independent pathway operating in parallel with host inflammasomes. Because IL-1ß function is maintained across species, yet cleavage by caspases does not appear to be, detection of microbial proteases may represent an ancestral system of innate immune regulation.

Salmonellae interactions with host processes.

Salmonellae invasion and intracellular replication within host cells result in a range of diseases, including gastroenteritis, bacteraemia, enteric fever and focal infections. In recent years, considerable progress has been made in our understanding of the molecular mechanisms that salmonellae use to alter host cell physiology; through the delivery of effector proteins with specific activities and through the modulation of defence and stress response pathways. In this Review, we summarize our current knowledge of the complex interplay between bacterial and host factors that leads to inflammation, disease and, in most cases, control of the infection by its animal hosts, with a particular focus on Salmonella enterica subsp. enterica serovar Typhimurium. We also highlight gaps in our knowledge of the contributions of salmonellae and the host to disease pathogenesis, and we suggest future avenues for further study.

Yersinia pestis insecticidal-like toxin complex (Tc) family proteins: characterization of expression, subcellular localization, and potential role in infection of the flea vector.

BACKGROUND: Toxin complex (Tc) family proteins were first identified as insecticidal toxins in Photorhabdus luminescens and have since been found in a wide range of bacteria. The genome of Yersinia pestis, the causative agent of bubonic plague, contains a locus that encodes the Tc protein homologues YitA, YitB, YitC, and YipA and YipB. Previous microarray data indicate that the Tc genes are highly upregulated by Y. pestis while in the flea vector; however, their role in the infection of fleas and pathogenesis in the mammalian host is unclear. RESULTS: We show that the Tc proteins YitA and YipA are highly produced by Y. pestis while in the flea but not during growth in brain heart infusion (BHI) broth at the same temperature. Over-production of the LysR-type regulator YitR from an exogenous plasmid increased YitA and YipA synthesis in broth culture. The increase in production of YitA and YipA correlated with the yitR copy number and was temperature-dependent. Although highly synthesized in fleas, deletion of the Tc proteins did not alter survival of Y. pestis in the flea or prevent blockage of the proventriculus. Furthermore, YipA was found to undergo post-translational processing and YipA and YitA are localized to the outer membrane of Y. pestis. YitA was also detected by immunofluorescence microscopy on the surface of Y. pestis. Both YitA and YipA are produced maximally at low temperature but persist for several hours after transfer to 37°C. CONCLUSIONS: Y. pestis Tc proteins are highly expressed in the flea but are not essential for Y. pestis to stably infect or produce a transmissible infection in the flea. However, YitA and YipA localize to the outer membrane and YitA is exposed on the surface, indicating that at least YitA is present on the surface when Y. pestis is transmitted into the mammalian host from the flea.

A Salmonella typhimurium-translocated glycerophospholipid:cholesterol acyltransferase promotes virulence by binding to the RhoA protein switch regions.

Salmonella enterica serovar typhimurium translocates a glycerophospholipid:cholesterol acyltransferase (SseJ) into the host cytosol after its entry into mammalian cells. SseJ is recruited to the cytoplasmic face of the host cell phagosome membrane where it is activated upon binding the small GTPase, RhoA. SseJ is regulated similarly to cognate eukaryotic effectors, as only the GTP-bound form of RhoA family members stimulates enzymatic activity. Using NMR and biochemistry, this work demonstrates that SseJ competes effectively with Rhotekin, ROCK, and PKN1 in binding to a similar RhoA surface. The RhoA surface that binds SseJ includes the regulatory switch regions that control activation of mammalian effectors. These data were used to create RhoA mutants with altered SseJ binding and activation. This structure-function analysis supports a model in which SseJ activation occurs predominantly through binding to residues within switch region II. We further defined the nature of the interaction between SseJ and RhoA by constructing SseJ mutants in the RhoA binding surface. These data indicate that SseJ binding to RhoA is required for recruitment of SseJ to the endosomal network and for full Salmonella virulence for inbred susceptible mice, indicating that regulation of SseJ by small GTPases is an important virulence strategy of this bacterial pathogen. The dependence of a bacterial effector on regulation by a mammalian GTPase defines further how intimately host pathogen interactions have coevolved through similar and divergent evolutionary strategies.

Activation of a bacterial virulence protein by the GTPase RhoA.

The Rho family of guanosine triphosphatases (GTPases) are essential eukaryotic signaling molecules that regulate cellular physiology. Virulence factors from various pathogens alter the signaling of GTPases by acting as GTPase activating factors, guanine nucleotide exchange factors, or direct covalent modifiers; however, bacterial virulence factors that sense rather than alter the signaling states of Rho GTPases have not been previously described. Here, we report that the translocated Salmonellae virulence factor SseJ binds to the guanosine triphosphate-bound form of RhoA. This interaction stimulates the lipase activity of SseJ, which results in the esterification of cholesterol in the host cell membrane. Our results suggest that the activation of molecules downstream of GTPases is not exclusive to eukaryotic proteins, and that a bacterial protein has evolved to recognize the activation state of RhoA, which regulates its enzymatic activity as part of the host-pathogen interaction.