Depleting CD103+ resident memory T cells in vivo reveals immunostimulatory functions in oral mucosa.

The oral mucosa is a frontline for microbial exposure and juxtaposes several unique tissues and mechanical structures. Based on parabiotic surgery of mice receiving systemic viral infections or co-housing with microbially diverse pet shop mice, we report that the oral mucosa harbors CD8+ CD103+ resident memory T cells (TRM), which locally survey tissues without recirculating. Oral antigen re-encounter during the effector phase of immune responses potentiated TRM establishment within tongue, gums, palate, and cheek. Upon reactivation, oral TRM triggered changes in somatosensory and innate immune gene expression. We developed in vivo methods for depleting CD103+ TRM while sparing CD103neg TRM and recirculating cells. This revealed that CD103+ TRM were responsible for inducing local gene expression changes. Oral TRM putatively protected against local viral infection. This study provides methods for generating, assessing, and in vivo depleting oral TRM, documents their distribution throughout the oral mucosa, and provides evidence that TRM confer protection and trigger responses in oral physiology and innate immunity.

Treatment Success Following Standard Antibiotic Treatment for Bacterial Vaginosis Is Not Associated With Pretreatment Genital Immune or Microbial Parameters.

Background: Bacterial vaginosis (BV) is a proinflammatory genital condition associated with adverse reproductive health outcomes, including increased HIV incidence. However, BV recurrence rates are high after standard antibiotic treatment. While the composition of the vaginal microbiota before BV treatment may be linked to BV recurrence, it is unclear whether the preceding genital immune milieu is predictive of treatment success. Methods: Here we assessed whether baseline vaginal soluble immune factors or the composition of the vaginal microbiota predicted treatment success 1 month after metronidazole treatment in 2 separate cohorts of women with BV, 1 in the United States and 1 in Kenya; samples within 48 hours of BV treatment were also available for the US cohort. Results: Neither soluble immune factors nor the composition of the vaginal microbiota before BV treatment was associated with treatment response in either cohort. In the US cohort, although the absolute abundances of key vaginal bacterial taxa pretreatment were not associated with treatment response, participants with sustained BV clearance had a more pronounced reduction in the absolute abundance of Gardnerella vaginalis immediately after treatment. Conclusions: Pretreatment immune and microbial parameters were not predictive of BV treatment success in these clinical cohorts.

Tissue-resident memory T cells trigger rapid exudation and local antibody accumulation.

Adaptive immunity is didactically partitioned into humoral and cell-mediated effector mechanisms, which may imply that each arm is separate and does not function together. Here, we report that the activation of CD8+ resident memory T cells (TRM) in nonlymphoid tissues triggers vascular permeability, which facilitates rapid distribution of serum antibodies into local tissues. TRM reactivation was associated with transcriptional upregulation of antiviral signaling pathways as well as Fc receptors and components of the complement cascade. Effects were local, but evidence is presented that TRM in brain and reproductive mucosa are both competent to induce rapid antibody exudation. TRM reactivation in the mouse female genital tract increased local concentrations of virus-specific neutralizing antibodies, including anti-vesicular stomatitis virus, and passively transferred anti-HIV antibodies. We showed that this response was sufficient to increase the efficacy of ex vivo vesicular stomatitis virus neutralization. These results indicate that CD8+ TRM antigen recognition can enhance local humoral immunity.

Changes in concentrations of cervicovaginal immune mediators across the menstrual cycle: a systematic review and meta-analysis of individual patient data.

BACKGROUND: Hormonal changes during the menstrual cycle play a key role in shaping immunity in the cervicovaginal tract. Cervicovaginal fluid contains cytokines, chemokines, immunoglobulins, and other immune mediators. Many studies have shown that the concentrations of these immune mediators change throughout the menstrual cycle, but the studies have often shown inconsistent results. Our understanding of immunological correlates of the menstrual cycle remains limited and could be improved by meta-analysis of the available evidence. METHODS: We performed a systematic review and meta-analysis of cervicovaginal immune mediator concentrations throughout the menstrual cycle using individual participant data. Study eligibility included strict definitions of the cycle phase (by progesterone or days since the last menstrual period) and no use of hormonal contraception or intrauterine devices. We performed random-effects meta-analyses using inverse-variance pooling to estimate concentration differences between the follicular and luteal phases. In addition, we performed a new laboratory study, measuring select immune mediators in cervicovaginal lavage samples. RESULTS: We screened 1570 abstracts and identified 71 eligible studies. We analyzed data from 31 studies, encompassing 39,589 concentration measurements of 77 immune mediators made on 2112 samples from 871 participants. Meta-analyses were performed on 53 immune mediators. Antibodies, CC-type chemokines, MMPs, IL-6, IL-16, IL-1RA, G-CSF, GNLY, and ICAM1 were lower in the luteal phase than the follicular phase. Only IL-1α, HBD-2, and HBD-3 were elevated in the luteal phase. There was minimal change between the phases for CXCL8, 9, and 10, interferons, TNF, SLPI, elafin, lysozyme, lactoferrin, and interleukins 1ß, 2, 10, 12, 13, and 17A. The GRADE strength of evidence was moderate to high for all immune mediators listed here. CONCLUSIONS: Despite the variability of cervicovaginal immune mediator measurements, our meta-analyses show clear and consistent changes during the menstrual cycle. Many immune mediators were lower in the luteal phase, including chemokines, antibodies, matrix metalloproteinases, and several interleukins. Only interleukin-1α and beta-defensins were higher in the luteal phase. These cyclical differences may have consequences for immunity, susceptibility to infection, and fertility. Our study emphasizes the need to control for the effect of the menstrual cycle on immune mediators in future studies.

Novel Lymphocytic Choriomeningitis Virus Strain Sustains Abundant Exhausted Progenitor CD8 T Cells without Systemic Viremia.

Lymphocytic choriomeningitis virus (LCMV) is the prototypic arenavirus and a natural mouse pathogen. LCMV-Armstrong, an acutely resolved strain, and LCMV-clone 13, a mutant that establishes chronic infection, have provided contrasting infection models that continue to inform the fundamental biology of T cell differentiation, regulation of exhaustion, and response to checkpoint blockade. In this study, we report the isolation and characterization of LCMV-Minnesota (LCMV-MN), which was naturally transmitted to laboratory mice upon cohousing with pet shop mice and shares 80-95% amino acid homology with previously characterized LCMV strains. Infection of laboratory mice with purified LCMV-MN resulted in viral persistence that was intermediate between LCMV-Armstrong and -clone 13, with widely disseminated viral replication and viremia that was controlled within 15-30 d, unless CD4 T cells were depleted prior to infection. LCMV-MN-responding CD8+ T cells biased differentiation toward the recently described programmed death-1 (PD-1)+CXCR5+Tim-3lo stemlike CD8+ T cell population (also referred to as progenitor exhausted T cells) that effectuates responses to PD-1 blockade checkpoint inhibition, a therapy that rejuvenates responses against chronic infections and cancer. This subset resembled previously characterized PD-1+TCF1+ stemlike CD8+ T cells by transcriptional, phenotypic, and functional assays, yet was atypically abundant. LCMV-MN may provide a tool to better understand the breadth of immune responses in different settings of chronic Ag stimulation as well as the ontogeny of progenitor exhausted T cells and the regulation of responsiveness to PD-1 blockade.

Cutting Edge: Nucleocapsid Vaccine Elicits Spike-Independent SARS-CoV-2 Protective Immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Neutralizing Abs target the receptor binding domain of the spike (S) protein, a focus of successful vaccine efforts. Concerns have arisen that S-specific vaccine immunity may fail to neutralize emerging variants. We show that vaccination with a human adenovirus type 5 vector expressing the SARS-CoV-2 nucleocapsid (N) protein can establish protective immunity, defined by reduced weight loss and viral load, in both Syrian hamsters and K18-hACE2 mice. Challenge of vaccinated mice was associated with rapid N-specific T cell recall responses in the respiratory mucosa. This study supports the rationale for including additional viral Ags in SARS-CoV-2 vaccines, even if they are not a target of neutralizing Abs, to broaden epitope coverage and immune effector mechanisms.

Nucleocapsid vaccine elicits spike-independent SARS-CoV-2 protective immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Neutralizing antibodies target the receptor binding domain of the spike (S) protein, a focus of successful vaccine efforts. Concerns have arisen that S-specific vaccine immunity may fail to neutralize emerging variants. We show that vaccination with HAd5 expressing the nucleocapsid (N) protein can establish protective immunity, defined by reduced weight loss and viral load, in both Syrian hamsters and k18-hACE2 mice. Challenge of vaccinated mice was associated with rapid N-specific T cell recall responses in the respiratory mucosa. This study supports the rationale for including additional viral antigens, even if they are not a target of neutralizing antibodies, to broaden epitope coverage and immune effector mechanisms.

Cutting Edge: Mouse SARS-CoV-2 Epitope Reveals Infection and Vaccine-Elicited CD8 T Cell Responses.

The magnitude of SARS-CoV-2-specific T cell responses correlates inversely with human disease severity, suggesting T cell involvement in primary control. Whereas many COVID-19 vaccines focus on establishing humoral immunity to viral spike protein, vaccine-elicited T cell immunity may bolster durable protection or cross-reactivity with viral variants. To better enable mechanistic and vaccination studies in mice, we identified a dominant CD8 T cell SARS-CoV-2 nucleoprotein epitope. Infection of human ACE2 transgenic mice with SARS-CoV-2 elicited robust responses to H2-Db/N219-227, and 40% of HLA-A*02+ COVID-19 PBMC samples isolated from hospitalized patients responded to this peptide in culture. In mice, i.m. prime-boost nucleoprotein vaccination with heterologous vectors favored systemic CD8 T cell responses, whereas intranasal boosting favored respiratory immunity. In contrast, a single i.v. immunization with recombinant adenovirus established robust CD8 T cell memory both systemically and in the respiratory mucosa.

Retrograde migration supplies resident memory T cells to lung-draining LN after influenza infection.

Numerous observations indicate that resident memory T cells (TRM) undergo unusually rapid attrition within the lung. Here we demonstrate that contraction of lung CD8+ T cell responses after influenza infection is contemporized with egress of CD69+/CD103+ CD8+ T cells to the draining mediastinal LN via the lymphatic vessels, which we term retrograde migration. Cells within the draining LN retained canonical markers of lung TRM, including CD103 and CD69, lacked Ly6C expression (also a feature of lung TRM), maintained granzyme B expression, and did not equilibrate among immunized parabiotic mice. Investigations of bystander infection or removal of the TCR from established memory cells revealed that the induction of the TRM phenotype was dependent on antigen recognition; however, maintenance was independent. Thus, local lung infection induces CD8+ T cells with a TRM phenotype that nevertheless undergo retrograde migration, yet remain durably committed to the residency program within the draining LN, where they provide longer-lived regional memory while chronicling previous upstream antigen experiences.

T cell-inducing vaccine durably prevents mucosal SHIV infection even with lower neutralizing antibody titers.

Recent efforts toward an HIV vaccine focus on inducing broadly neutralizing antibodies, but eliciting both neutralizing antibodies (nAbs) and cellular responses may be superior. Here, we immunized macaques with an HIV envelope trimer, either alone to induce nAbs, or together with a heterologous viral vector regimen to elicit nAbs and cellular immunity, including CD8+ tissue-resident memory T cells. After ten vaginal challenges with autologous virus, protection was observed in both vaccine groups at 53.3% and 66.7%, respectively. A nAb titer >300 was generally associated with protection but in the heterologous viral vector + nAb group, titers <300 were sufficient. In this group, protection was durable as the animals resisted six more challenges 5 months later. Antigen stimulation of T cells in ex vivo vaginal tissue cultures triggered antiviral responses in myeloid and CD4+ T cells. We propose that cellular immune responses reduce the threshold of nAbs required to confer superior and durable protection.

Impact of Endemic Infections on HIV Susceptibility in Sub-Saharan Africa.

Human immunodeficiency virus (HIV) remains a leading cause of global morbidity with the highest burden in Sub-Saharan Africa (SSA). For reasons that are incompletely understood, the likelihood of HIV transmission is several fold higher in SSA than in higher income countries, and most of these infections are acquired by young women. Residents of SSA are also exposed to a variety of endemic infections, such as malaria and various helminthiases that could influence mucosal and systemic immunology. Since these immune parameters are important determinants of HIV acquisition and progression, this review explores the possible effects of endemic infections on HIV susceptibility and summarizes current knowledge of the epidemiology and underlying immunological mechanisms by which endemic infections could impact HIV acquisition. A better understanding of the interaction between endemic infections and HIV may enhance HIV prevention programs in SSA.

Protocol for a randomized clinical trial exploring the effect of antimicrobial agents on the penile microbiota, immunology and HIV susceptibility of Ugandan men.

BACKGROUND: The foreskin is the main site of HIV acquisition in a heterosexual uncircumcised man, but many men in endemic countries are reluctant to undergo penile circumcision (PC). Observational studies suggest that proinflammatory anaerobic bacteria are enriched on the uncircumcised penis, where they may enhance HIV susceptibility through increased foreskin inflammatory cytokines and the recruitment of HIV-susceptible CD4+ target cells. This trial will examine the impact of systemic and topical antimicrobials on ex vivo foreskin HIV susceptibility. METHODS/DESIGN: This randomized, open-label clinical trial will randomize 125 HIV-negative Ugandan men requesting voluntary PC to one of five arms (n = 25 each). The control group will receive immediate PC, while the four intervention groups will defer PC for 1 month and be provided in the interim with either oral tinidazole, penile topical metronidazole, topical clindamycin, or topical hydrogen peroxide. The impact of these interventions on HIV entry into foreskin-derived CD4+ T cells will be quantified ex vivo at the time of PC using a clade A, R5 tropic HIV pseudovirus assay (primary endpoint); secondary endpoints include the impact of antimicrobials on immune parameters and the microbiota of the participant's penis and of the vagina of their female partner (if applicable), assessed by multiplex enzyme-linked immunosorbent assay and 16S rRNA sequencing. DISCUSSION: There is a critical need to develop acceptable, simple, and effective means of HIV prevention in men unwilling to undergo PC. This trial will provide insight into the causative role of the foreskin microbiota on HIV susceptibility, and the impact of simple microbiota-focused clinical interventions. This may pave the way for future clinical trials using low-cost, nonsurgical intervention(s) to reduce HIV risk in uncircumcised heterosexual men. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03412071 . Retrospectively registered on 26 January 2018.

Characterization of CD4+ T cell subsets and HIV susceptibility in the inner and outer foreskin of Ugandan men.

PROBLEM: Biological mechanisms of foreskin HIV acquisition are poorly defined. The inner foreskin is preferentially infected in explant models, so we hypothesized that this site would be enriched for HIV-susceptible CD4+ T cells and proinflammatory/chemoattractant cytokines. METHOD OF STUDY: A total of 42 HIV-uninfected Ugandan men without genital symptoms provided foreskin tissues and swabs at the time of elective penile circumcision. The immune phenotype of foreskin-derived CD4+ T cells and entry of a CCR5-tropic HIV pseudovirus was characterized, and specific cytokine levels assayed by multiplexed chemiluminescent ELISA. RESULTS: Unexpectedly, outer foreskin CD4+ T cells more frequently expressed CCR5 (median 29.2% vs 22.9%, P = 0.01) and CD69 (median 36.5% vs 15%, P < 0.01), and on a per-cell basis, HIV entry was higher. However, overall CD4+ T cell density was approximately twofold higher in the inner foreskin, and several highly susceptible T cell subsets were increased at this site, including Th17 cells (20.0% vs 14.1%, P = 0.0021). Specific pro-inflammatory cytokine levels were also higher on the inner foreskin surface (IL-17, IL-8, RANTES and IL-1ß; all P < 0.05). CONCLUSION: There was marked heterogeneity in CD4+ T cell populations and immune milieu between inner and outer foreskin tissues. Despite higher per-cell viral entry into CD4+ T cells from the outer foreskin, the higher target cell density and enriched pro-inflammatory cytokines of the inner foreskin suggest that this may be a preferential site for HIV acquisition.

Schistosoma mansoni treatment reduces HIV entry into cervical CD4+ T cells and induces IFN-I pathways.

Schistosoma mansoni (Sm) infection has been linked with an increased risk of HIV acquisition in women. Therefore, defining the mechanism(s) by which Sm alters HIV susceptibility might lead to new HIV prevention strategies. Here, we analyze the impact of standard Sm therapy in HIV-uninfected Sm+ Ugandan adult women on genital HIV susceptibility and mucosal and systemic immunology. Schistosomiasis treatment induces a profound reduction of HIV entry into cervical and blood CD4+ T cells that is sustained for up to two months, despite transient systemic and mucosal immune activation and elevated genital IL-1α levels. Genital IFN-α2a levels are also elevated post-treatment, and IFN-α2a blocks HIV entry into primary CD4+ T cells ex vivo. Transcriptomic analysis of blood mononuclear cells post-Sm treatment shows IFN-I pathway up-regulation and partial reversal of Sm-dysregulated interferon signaling. These findings indicate that Sm therapy may reduce HIV susceptibility for women with Sm infection, potentially through de-repression of IFN-I pathways.

Impact of Standard Bacterial Vaginosis Treatment on the Genital Microbiota, Immune Milieu, and Ex Vivo Human Immunodeficiency Virus Susceptibility.

BACKGROUND: Genital immunology is a key determinant of human immunodeficiency virus (HIV) susceptibility. Both factors are modulated by bacterial vaginosis (BV) and, to some extent, by Lactobacillus iners, the genital Lactobacillus spp. that predominates in African, Caribbean, and other Black (ACB) women. We conducted a clinical trial to assess the impact of oral metronidazole treatment on the genital immune parameters of HIV acquisition risks in Kenyan women with BV. METHODS: The primary endpoint was ex vivo cervical CD4+ T-cell HIV susceptibility after 1 month; secondary endpoints included genital cytokine/chemokine levels, cervical immune cell populations, and the composition of the cervico-vaginal microbiota by 16S ribosomal RNA gene amplicon sequencing. RESULTS: BV resolved (Nugent score ≤ 3) at 1 month in 20/45 participants, and cervical CD4+ T-cell HIV entry was moderately reduced in all participants, regardless of treatment outcome. Resolution of BV and reduced abundances of BV-associated gram-negative taxa correlated with reduced genital interleukin (IL)-1α/ß. However, BV resolution and the concomitant colonization by Lactobacillus iners substantially increased several genital chemokines associated with HIV acquisition, including interferon-γ inducible protein (IP)-10, macrophage inflammatory protein (MIP)-3α, and monokine induced by gamma interferon (MIG). In an independent cohort of ACB women, most of whom were BV-free, vaginal chemokines were again closely linked with L. iners abundance, though not other Lactobacillus spp. CONCLUSIONS: BV treatment reduced genital CD4+ T-cell HIV susceptibility and IL-1 levels, but dramatically increased the genital chemokines that may enhance HIV susceptibility; the latter effect was related to the restoration of an Lactobacillus iners-dominated microbiota. Further studies are needed before treatment of asymptomatic BV can be recommended for HIV prevention in ACB communities.

Ex vivo HIV entry into blood CD4+ T cells does not predict heterosexual HIV acquisition in women.

BACKGROUND: A blood-based assay that could quantify HIV susceptibility would be very valuable for HIV prevention research. Previously, we developed and validated an ex vivo, flow-based, HIV entry assay to assess genital HIV susceptibility in endocervical CD4+ T cells. METHODS: Here we assessed whether this tool could be used to predict HIV risk using blood-derived CD4+ T cells in a rigorously-blinded, nested case-control study using blood samples collected from high-risk, HIV-uninfected South African women enrolled in the CAPRISA 004 clinical trial. Cases, subsequently acquiring HIV were sampled prior to HIV infection and compared with controls, who remained HIV-uninfected. The primary endpoint was ex vivo entry of a CCR5-tropic HIV founder virus into blood CD4+ T cells. Secondary endpoints included HIV entry into CD4+ central (TCM) and effector (TEM) memory T cells, and into CD4+ T cell subsets expressing CCR5, CD69, CCR6, α4ß1 or α4ß7. RESULTS: Compared to bulk CD4+ T cells (4.9% virus entry), CD4+ T cells expressing CCR5, CCR6 or α4ß1 and TEM were highly susceptible (15.5%, 8.8%, 8.2% and 10.8% entry, respectively, all p<0.0001), while TCM, CD69+ or α4ß7+ CD4+ cells were moderately susceptible (6.4%, 6.0% and 5.8% respectively, p ≤ 0.003). While the proportion of the aforementioned highly susceptible cells correlated with overall virus entry into CD4+ T cells within an individual (r = 0.68, 0.47, 0.67, and 0.60 respectively, p<0.0001), blood virus entry did not predict subsequent mucosal HIV acquisition after controlling for sexual behaviour and condom use (OR 0.92, 95% CI 0.77-1.11, p = 0.40). CONCLUSIONS: Although virus entry identified several previously known highly susceptible cellular HIV targets, blood HIV entry did not predict subsequent heterosexual HIV acquisition. Assessment of mucosal HIV susceptibility may require sampling at the site of HIV exposure.

Integrin α4ß7 expression on peripheral blood CD4+ T cells predicts HIV acquisition and disease progression outcomes.

The gastrointestinal (GI) mucosa is central to HIV pathogenesis, and the integrin α4ß7 promotes the homing of immune cells to this site, including those that serve as viral targets. Data from simian immunodeficiency virus (SIV) animal models suggest that α4ß7 blockade provides prophylactic and therapeutic benefits. We show that pre-HIV infection frequencies of α4ß7+ peripheral blood CD4+ T cells, independent of other T cell phenotypes and genital inflammation, were associated with increased rates of HIV acquisition in South African women. A similar acquisition effect was observed in a Kenyan cohort and in nonhuman primates (NHPs) after intravaginal SIV challenge. This association was stronger when infection was caused by HIV strains containing V2 envelope motifs with a preference for α4ß7 binding. In addition, pre-HIV α4ß7+ CD4+ T cells predicted a higher set-point viral load and a greater than twofold increased rate of CD4+ T cell decline. These results were confirmed in SIV-infected NHPs. Increased frequencies of pre-HIV α4ß7+ CD4+ T cells were also associated with higher postinfection expression of lipopolysaccharide binding protein, a microbial translocation marker, suggestive of more extensive gut damage. CD4+ T cells expressing α4ß7 were rapidly depleted very early in HIV infection, particularly from the GI mucosa, and were not restored by early antiretroviral therapy. This study provides a link between α4ß7 expression and HIV clinical outcomes in humans, in line with observations made in NHPs. Given the availability of a clinically approved anti-α4ß7 monoclonal antibody for treatment of inflammatory bowel disease, these data support further evaluation of targeting α4ß7 integrin as a clinical intervention during HIV infection.

Inflammation and HIV Transmission in Sub-Saharan Africa.

While the per-contact risk of sexual HIV transmission is relatively low, it is fourfold higher in sub-Saharan Africa, and this may partly explain the major global disparities that exist in HIV prevalence. Genital immune parameters are key determinants of HIV transmission risk, including epithelial integrity and the presence of highly HIV-susceptible intraepithelial or submucosal CD4+ T cell target cells. Biological parameters that may enhance mucosal HIV susceptibility in highly HIV-affected regions of sub-Saharan Africa include increased levels of mucosal inflammation, which can affect both epithelial integrity and target cell availability, as well as the increased mucosal surface area that is afforded by an intact foreskin, contraceptive choices, and intravaginal practices. There are multifactorial causes for increased mucosal inflammation, with the prevalence and nature of common co-infections being particularly relevant.

Proteolytic processing of Nlrp1b is required for inflammasome activity.

Nlrp1b is a NOD-like receptor that detects the catalytic activity of anthrax lethal toxin and subsequently co-oligomerizes into a pro-caspase-1 activation platform known as an inflammasome. Nlrp1b has two domains that promote oligomerization: a NACHT domain, which is a member of the AAA+ ATPase family, and a poorly characterized Function to Find Domain (FIIND). Here we demonstrate that proteolytic processing within the FIIND generates N-terminal and C-terminal cleavage products of Nlrp1b that remain associated in both the auto-inhibited state and in the activated state after cells have been treated with lethal toxin. Functional significance of cleavage was suggested by the finding that mutations that block processing of Nlrp1b also prevent the ability of Nlrp1b to activate pro-caspase-1. By using an uncleaved mutant of Nlrp1b, we established the importance of cleavage by inserting a heterologous TEV protease site into the FIIND and demonstrating that TEV protease processed this site and induced inflammasome activity. Proteolysis of Nlrp1b was shown to be required for the assembly of a functional inflammasome: a mutation within the FIIND that abolished cleavage had no effect on self-association of a FIIND-CARD fragment, but did reduce the recruitment of pro-caspase-1. Our work indicates that a post-translational modification enables Nlrp1b to function.

Rapid autocatalytic activation of the M4 metalloprotease aureolysin is controlled by a conserved N-terminal fungalysin-thermolysin-propeptide domain.

SUMMARY: The Staphylococcus aureus proteolytic cascade consists of a metalloprotease aureolysin (Aur), which activates a serine protease zymogen proSspA, which in turn activates the SspB cysteine protease. As with other M4 metalloproteases, including elastase of Pseudomonas aeruginosa, the propeptide of proAur contains an N-terminal fungalysin-thermolysin-propeptide (FTP) domain. Autocatalytic activation of proAur was initiated by processing at T85 downward arrowL(86) in the FTP domain. This differed from the mechanism described for proElastase, where the FTP domain has an RY motif in place of TL(86), and processing occurred at the junction of the propeptide and metalloprotease domains, which remained as an inactive complex during passage across the outer membrane. When TL(86) in the FTP domain was replaced with RY, an intact N-terminal propeptide was secreted, but the M4 metalloprotease domain was degraded. Consequently, this segment of the FTP domain promotes intramolecular processing of proAur while bestowing a chaperone function, but discourages processing within the FTP domain of proElastase, where activation must be co-ordinated with passage across a second membrane. We conclude that the FTP domain of proAur is adapted to facilitate a rapid autocatalytic activation mechanism, consistent with the role or proAur as initiator of the staphylococcal proteolytic cascade.