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

Chemical Optimization of Selective Pseudomonas aeruginosa LasB Elastase Inhibitors and Their Impact on LasB-Mediated Activation of IL-1β 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-1 beta. Furthermore, inhibition of LasBmediated IL-1 beta 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

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

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-1β 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-1β 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

Mouse IL-18 can be activated by SpeB but is not secreted under normal inert conditions.

(A) C57BL/6 wild-type or IL-18-knockout (il18-/-) mice were inoculated intradermally with 108 CFU of GAS 5448 or its DspeB mutant. After 72 h, mice were euthanized, and GAS CFU was enumerated at the infection site. Results are from 2 independent experiments with 5 mice in each. (B) Recombinant mouse pro-IL-18 was incubated with human Caspase-1 or SpeB and activation measured with HEK-Blue IL18 reporter cells. (C) Supernatants or lysates from mouse primary keratinocytes were examined for IL-18 by ELISA and (D) cell lysis confirmed by LDH release assay, or (E) incubated 4 h with SpeB, and active IL-18 was quantified with HEK-Blue IL-18 reporter cells. Data represent at least 3 independent experiments with 4 replicates. Data were analyzed by 1-way ANOVA using Dunnett multiple comparisons analysis. Bars show median values ± standard deviation. *P P <0.005; ns, not significant.</p

Cytokine profiles of keratinocytes and related cell lines.

HaCaT, Detroit 562, HEp-2, A-431, primary keratinocytes, or HUVEC cells, were infected with 7.5 x 106 colony-forming units (CFU) of GAS for 6 h. (A) Relative abundance of select cytokines was examined by membrane-based antibody array. (B) Cytokine profiles of each cell were examined by multivariate (principal component analysis), of the total variance, PC1 explains 48.67% and PC2 20.87%, from the raw cytokine quantities tabulated in (A). Arrows indicate change in cells from uninfected to 6 h infection. (C) Graphical representation of the congruent cytokine profiles between cell types.</p

Examination of GAS requirements for IL-18 activation.

Primary human keratinocytes were infected with GAS at MOI 10 for 4 h, then (A, B) bioactive IL-18 was measured with HEK-Blue IL18 reporter cells. (C) SpeB activity was measured using specific substrate sub103 and IL-18 activity measured in the supernatants from keratinocytes treated with titrations of purified SpeB protein. (D) Recombinant human pro-IL-18 was purified and incubated with purified, active SpeB, then cleavage products were separated by SDS-PAGE and visualized by staining. (E) Coding sequence of human IL-18 with probable and known cleavage sites indicated; the largest and smallest confirmed by Edman sequencing. Data were analyzed by 1-way ANOVA using Dunnett multiple comparisons analysis. All data represent at least 3 independent experiments with 4 replicates. Bars show median values ± standard deviation. **P <0.005; ns, not significant.</p

SpeB activation of IL-18 promotes antimicrobial IFN-γ responses.

(A) Diagram of primary keratinocyte/PBMC co-culture model; IFN-γ is a reporter of T cell activation, which can occur via IL-18 and additional mechanisms during infection. (B) Cocultured keratinocyte/PBMCs were infected with 105 CFU of GAS or treated with rSpeB. After 4 h, active IL-18 was quantified with HEK-Blue IL-18 reporter cells and IFN-γ by ELISA. (C, D) Cocultured cells were treated with SpeB as in (B), in combination or post-depletion of CD2+ T cells, or with treatments with IL-18 neutralizing antibody or 5 μM YVAD-cmk or 10 μM VX765 to inhibit inflammasome function. Data represent at least 3 independent experiments with 4 replicates. Data were analyzed by 1-way ANOVA using Dunnett multiple comparisons analysis. Bars show median values ± standard deviation. *P P <0.005; ns, not significant.</p

Bacterial activation of IL-18.

(A, B) IL-18 activation was measured in the supernatants from keratinocytes infected with the indicated gene knockouts of GAS strain 5448 or wild-type GAS, and after 4 h, bioactive IL-18 was measured with HEK-Blue IL18 reporter cells. (C) IL-18 activation was measured in the supernatants from keratinocytes infected with the indicated bacterial species and after 4 h, bioactive IL-18 was measured with HEK-Blue IL18 reporter cells. (D, E) IL-18 activation was measured in the supernatants from keratinocytes infected with the indicated gene knockouts of GAS strain 5448, L. lactis, or during treatment with 5 μM E-64. Spectinomycin and anhydrotetracycline to maintain SpeB expression from the indicated plasmids. Data represent at least 3 independent experiments with 4 replicates. Data were analyzed by 1-way ANOVA using Dunnett multiple comparisons analysis compared to uninfected/untreated keratinocytes. Bars show median values ± standard deviation. *P P <0.005; ns, not significant.</p

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