Neonatal Sepsis Due to Multidrug-resistant Bacteria at a Tertiary Teaching Hospital in Ethiopia.

BACKGROUND: The burden of multidrug-resistant bacterial infections in low-income countries is alarming. This study aimed to identify the bacterial etiologies and antibiotic resistance patterns among neonates in Jimma, Ethiopia. METHODS: An observational longitudinal study was conducted among 238 presumptive neonatal sepsis cases tested with blood and/or cerebrospinal fluid culture. The bacterial etiologies were confirmed using matrix-assisted laser desorption ionization-time of flight mass spectrometry. The antibiotic resistance patterns were determined using the automated disc diffusion method (Bio-Rad) and the results were interpreted based on the European Committee on Antimicrobial Susceptibility Testing 2021 breakpoints. Extended-spectrum ß-lactamases were detected using a double disc synergy test and confirmed by Mast discs (Mast Diagnostica GmbH). RESULTS: A total of 152 pathogens were identified. Of these, Staphylococcus aureus (18.4%) was the predominant isolate followed by Klebsiella pneumoniae (15.1%) and Escherichia coli (10.5%). All the isolates exhibited a high rate of resistance to first- and second-line antibiotics ranging from 73.3% for gentamicin to 93.3% for ampicillin. Furthermore, 74.4% of the Gram-negative isolates were extended-spectrum ß-lactamase producers and 57.1% of S. aureus strains were methicillin resistant. The case fatality rate was 10.1% and 66.7% of the deaths were attributable to infections by multidrug-resistant pathogens. CONCLUSIONS: The study revealed a high rate of infections with multidrug-resistant pathogens. This poses a significant challenge to the current global and national target to reduce neonatal mortality rates. To address these challenges, it is important to employ robust infection prevention practices and continuous antibiotic resistance testing to allow targeted therapy.

Hospital Wastes as Potential Sources for Multi-Drug-Resistant ESBL-Producing Bacteria at a Tertiary Hospital in Ethiopia.

The hospital environment is increasingly becoming an important reservoir for multi-drug-resistant (MDR) Gram-negative bacteria, posing serious challenges to efforts to combat antimicrobial resistance (AMR). This study aimed to investigate the role of hospital waste as a potential source of MDR ESBL-producing bacteria. Samples were collected from multiple sources within a hospital and its vicinity, including surface swabs, houseflies, and sewage samples. The samples were subsequently processed in a microbiology laboratory to identify potential pathogenic bacteria and confirmed using MALDI-TOF MS. Bacteria were isolated from 87% of samples, with the predominant isolates being E. coli (30.5%), Klebsiella spp. (12.4%), Providencia spp. (12.4%), and Proteus spp. (11.9%). According to the double disc synergy test (DDST) analysis, nearly half (49.2%) of the bacteria were identified as ESBL producers. However, despite exhibiting complete resistance to beta-lactam antibiotics, 11.8% of them did not test positive for ESBL production. The characterization of E. coli revealed that 30.6% and 5.6% of them carried blaCTX-M group 1 type-15 and blaNDM genes, respectively. This finding emphasizes the importance of proper hospital sanitation and waste management practices to mitigate the spread of AMR within the healthcare setting and safeguard the health of both patients and the wider community.

Molecular characterization of carbapenem-resistance in Gram-negative isolates obtained from clinical samples at Jimma Medical Center, Ethiopia.

Background: In resource-constrained settings, limited antibiotic options make treating carbapenem-resistant bacterial infections difficult for healthcare providers. This study aimed to assess carbapenemase expression in Gram-negative bacteria isolated from clinical samples in Jimma, Ethiopia. Methods: A cross-sectional study was conducted to assess carbapenemase expression in Gram-negative bacteria isolated from patients attending Jimma Medical Center. Totally, 846 Gram-negative bacteria were isolated and identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Phenotypic antibiotic resistance patterns were determined using the Kirby-Bauer disk diffusion method and Etest strips. Extended-spectrum ß-lactamase phenotype was determined using MAST disks, and carbapenemases were characterized using multiplex polymerase chain reactions (PCR). Results: Among the isolates, 19% (157/846) showed phenotypic resistance to carbapenem antibiotics. PCR analysis revealed that at least one carbapenemase gene was detected in 69% (107/155) of these strains. The most frequently detected acquired genes were blaNDM in 35% (37/107), blaVIM in 24% (26/107), and blaKPC42 in 13% (14/107) of the isolates. Coexistence of two or more acquired genes was observed in 31% (33/107) of the isolates. The most common coexisting acquired genes were blaNDM + blaOXA-23, detected in 24% (8/33) of these isolates. No carbapenemase-encoding genes could be detected in 31% (48/155) of carbapenem-resistant isolates, with P. aeruginosa accounting for 85% (41/48) thereof. Conclusion: This study revealed high and incremental rates of carbapenem-resistant bacteria in clinical samples with various carbapenemase-encoding genes. This imposes a severe challenge to effective patient care in the context of already limited treatment options against Gram-negative bacterial infections in resource-constrained settings.

Translational Studies Using the MALT1 Inhibitor (S)-Mepazine to Induce Treg Fragility and Potentiate Immune Checkpoint Therapy in Cancer.

Introduction: Regulatory T cells (Tregs) play a critical role in the maintenance of immune homeostasis but also protect tumors from immune-mediated growth control or rejection and pose a significant barrier to effective immunotherapy. Inhibition of MALT1 paracaspase activity can selectively reprogram immune-suppressive Tregs in the tumor microenvironment to adopt a proinflammatory fragile state, which offers an opportunity to impede tumor growth and enhance the efficacy of immune checkpoint therapy (ICT). Methods: We performed preclinical studies with the orally available allosteric MALT1 inhibitor (S)-mepazine as a single-agent and in combination with anti-programmed cell death protein 1 (PD-1) ICT to investigate its pharmacokinetic properties and antitumor effects in several murine tumor models as well as patient-derived organotypic tumor spheroids (PDOTS). Results: (S)-mepazine demonstrated significant antitumor effects and was synergistic with anti-PD-1 therapy in vivo and ex vivo but did not affect circulating Treg frequencies in healthy rats at effective doses. Pharmacokinetic profiling revealed favorable drug accumulation in tumors to concentrations that effectively blocked MALT1 activity, potentially explaining preferential effects on tumor-infiltrating over systemic Tregs. Conclusions: The MALT1 inhibitor (S)-mepazine showed single-agent anticancer activity and presents a promising opportunity for combination with PD-1 pathway-targeted ICT. Activity in syngeneic tumor models and human PDOTS was likely mediated by induction of tumor-associated Treg fragility. This translational study supports ongoing clinical investigations (ClinicalTrials.gov Identifier: NCT04859777) of MPT-0118, (S)-mepazine succinate, in patients with advanced or metastatic treatment-refractory solid tumors.

TRAF6 controls T cell homeostasis by maintaining the equilibrium of MALT1 scaffolding and protease functions.

MALT1 is a core component of the CARD11-BCL10-MALT1 (CBM) signalosome, in which it acts as a scaffold and a protease to bridge T cell receptor (TCR) ligation to immune activation. As a scaffold, MALT1 binds to TRAF6, and T cell-specific TRAF6 ablation or destruction of MALT1-TRAF6 interaction provokes activation of conventional T (Tconv) effector cells. In contrast, MALT1 protease activity controls the development and suppressive function of regulatory T (Treg) cells in a T cell-intrinsic manner. Thus, complete loss of TRAF6 or selective inactivation of MALT1 catalytic function in mice skews the immune system towards autoimmune inflammation, but distinct mechanisms are responsible for these immune disorders. Here we demonstrate that TRAF6 deletion or MALT1 paracaspase inactivation are highly interdependent in causing the distinct immune pathologies. We crossed mice with T cell-specific TRAF6 ablation (Traf6-ΔT) and mice with a mutation rendering the MALT1 paracaspase dead in T cells (Malt1 PD-T) to yield Traf6-ΔT;Malt1 PD-T double mutant mice. These mice reveal that the autoimmune inflammation caused by TRAF6-ablation relies strictly on the function of the MALT1 protease to drive the activation of Tconv cells. Vice versa, despite the complete loss of Treg cells in Traf6-ΔT;Malt1 PD-T double mutant mice, inactivation of the MALT1 protease is unable to cause autoinflammation, because the Tconv effector cells are not activated in the absence of TRAF6. Consequentially, combined MALT1 paracaspase inactivation and TRAF6 deficiency in T cells mirrors the immunodeficiency seen upon T cell-specific MALT1 ablation.

Human TH17 cells engage gasdermin E pores to release IL-1α on NLRP3 inflammasome activation.

It has been shown that innate immune responses can adopt adaptive properties such as memory. Whether T cells utilize innate immune signaling pathways to diversify their repertoire of effector functions is unknown. Gasdermin E (GSDME) is a membrane pore-forming molecule that has been shown to execute pyroptotic cell death and thus to serve as a potential cancer checkpoint. In the present study, we show that human T cells express GSDME and, surprisingly, that this expression is associated with durable viability and repurposed for the release of the alarmin interleukin (IL)-1α. This property was restricted to a subset of human helper type 17 T cells with specificity for Candida albicans and regulated by a T cell-intrinsic NLRP3 inflammasome, and its engagement of a proteolytic cascade of successive caspase-8, caspase-3 and GSDME cleavage after T cell receptor stimulation and calcium-licensed calpain maturation of the pro-IL-1α form. Our results indicate that GSDME pore formation in T cells is a mechanism of unconventional cytokine release. This finding diversifies our understanding of the functional repertoire and mechanistic equipment of T cells and has implications for antifungal immunity.

Phosphorylation of serine-893 in CARD11 suppresses the formation and activity of the CARD11-BCL10-MALT1 complex in T and B cells.

CARD11 acts as a gatekeeper for adaptive immune responses after T cell or B cell antigen receptor (TCR/BCR) ligation on lymphocytes. PKCθ/ß-catalyzed phosphorylation of CARD11 promotes the assembly of the CARD11-BCL10-MALT1 (CBM) complex and lymphocyte activation. Here, we demonstrated that PKCθ/ß-dependent CARD11 phosphorylation also suppressed CARD11 functions in T or B cells. Through mass spectrometry-based proteomics analysis, we identified multiple constitutive and inducible CARD11 phosphorylation sites in T cells. We demonstrated that a single TCR- or BCR-inducible phosphorylation on Ser893 in the carboxyl terminus of CARD11 prevented the activation of the transcription factor NF-κB, the kinase JNK, and the protease MALT1. Moreover, CARD11 Ser893 phosphorylation sensitized BCR-addicted lymphoma cells to toxicity induced by Bruton's tyrosine kinase (BTK) inhibitors. Phosphorylation of Ser893 in CARD11 by PKCθ controlled the strength of CARD11 scaffolding by impairing the formation of the CBM complex. Thus, PKCθ simultaneously catalyzes both stimulatory and inhibitory CARD11 phosphorylation events, which shape the strength of CARD11 signaling in lymphocytes.

A20 and ABIN-1 cooperate in balancing CBM complex-triggered NF-κB signaling in activated T cells.

T cell activation initiates protective adaptive immunity, but counterbalancing mechanisms are critical to prevent overshooting responses and to maintain immune homeostasis. The CARD11-BCL10-MALT1 (CBM) complex bridges T cell receptor engagement to NF-κB signaling and MALT1 protease activation. Here, we show that ABIN-1 is modulating the suppressive function of A20 in T cells. Using quantitative mass spectrometry, we identified ABIN-1 as an interactor of the CBM signalosome in activated T cells. A20 and ABIN-1 counteract inducible activation of human primary CD4 and Jurkat T cells. While A20 overexpression is able to silence CBM complex-triggered NF-κB and MALT1 protease activation independent of ABIN-1, the negative regulatory function of ABIN-1 depends on A20. The suppressive function of A20 in T cells relies on ubiquitin binding through the C-terminal zinc finger (ZnF)4/7 motifs, but does not involve the deubiquitinating activity of the OTU domain. Our mechanistic studies reveal that the A20/ABIN-1 module is recruited to the CBM complex via A20 ZnF4/7 and that proteasomal degradation of A20 and ABIN-1 releases the CBM complex from the negative impact of both regulators. Ubiquitin binding to A20 ZnF4/7 promotes destructive K48-polyubiquitination to itself and to ABIN-1. Further, after prolonged T cell stimulation, ABIN-1 antagonizes MALT1-catalyzed cleavage of re-synthesized A20 and thereby diminishes sustained CBM complex signaling. Taken together, interdependent post-translational mechanisms are tightly controlling expression and activity of the A20/ABIN-1 silencing module and the cooperative action of both negative regulators is critical to balance CBM complex signaling and T cell activation.

TRAF6 prevents fatal inflammation by homeostatic suppression of MALT1 protease.

Balanced control of T cell signaling is critical for adaptive immunity and protection from autoimmunity. By combining genetically engineered mouse models, biochemical analyses and pharmacological interventions, we describe an unexpected dual role of the tumor necrosis factor receptor­associated factor 6 (TRAF6) E3 ligase as both a positive and negative regulator of mucosa-associated lymphoid tissue 1 (MALT1) paracaspase. Although MALT1-TRAF6 recruitment is indispensable for nuclear factor κB signaling in activated T cells, TRAF6 counteracts basal MALT1 protease activity in resting T cells. In mice, loss of TRAF6-mediated homeostatic suppression of MALT1 protease leads to severe autoimmune inflammation, which is completely reverted by genetic or therapeutic inactivation of MALT1 protease function. Thus, TRAF6 functions as a molecular brake for MALT1 protease in resting T cells and a signaling accelerator for MALT1 scaffolding in activated T cells, revealing that TRAF6 controls T cell activation in a switch-like manner. Our findings have important implications for development and treatment of autoimmune diseases.

Use of Non-Natural Amino Acids for the Design and Synthesis of a Selective, Cell-Permeable MALT1 Activity-Based Probe.

Constitutive proteolytic activity of MALT1 is associated with highly aggressive B-cell lymphomas. Chemical tools that detect active MALT1 have been reported, but suffer from poor cell permeability and/or cross-reactivity with the cysteine protease cathepsin B. Here, we report that the non-natural amino acid pipecolinic acid in the P2 position of substrates and chemical probes leads to improved selectivity toward MALT1 and results in cell-permeable fluorescent probes.

Hectd3 promotes pathogenic Th17 lineage through Stat3 activation and Malt1 signaling in neuroinflammation.

Polyubiquitination promotes proteasomal degradation, or signaling and localization, of targeted proteins. Here we show that the E3 ubiquitin ligase Hectd3 is necessary for pathogenic Th17 cell generation in experimental autoimmune encephalomyelitis (EAE), a mouse model for human multiple sclerosis. Hectd3-deficient mice have lower EAE severity, reduced Th17 program and inefficient Th17 cell differentiation. However, Stat3, but not RORγt, has decreased polyubiquitination, as well as diminished tyrosine-705 activating phosphorylation. Additionally, non-degradative polyubiquitination of Malt1, critical for NF-κB activation and Th17 cell function, is reduced. Mechanistically, Hectd3 promotes K27-linked and K29-linked polyubiquitin chains on Malt1, and K27-linked polyubiquitin chains on Stat3. Moreover, Stat3 K180 and Malt1 K648 are targeted by Hectd3 for non-degradative polyubiquitination to mediate robust generation of RORγt+IL-17Ahi effector CD4+ T cells. Thus, our studies delineate a mechanism connecting signaling related polyubiquitination of Malt1 and Stat3, leading to NF-kB activation and RORγt expression, to pathogenic Th17 cell function in EAE.

BCL10-CARD11 Fusion Mimics an Active CARD11 Seed That Triggers Constitutive BCL10 Oligomerization and Lymphocyte Activation.

Assembly of the CARD11/CARMA1-BCL10-MALT1 (CBM) signaling complex upon T or B cell antigen receptor (TCR or BCR) engagement drives lymphocyte activation. Recruitment of pre-assembled BCL10-MALT1 complexes to CARD11 fosters activation of the MALT1 protease and canonical NF-κB signaling. Structural data and in vitro assays have suggested that CARD11 acts as a seed that nucleates the assembly of BCL10 filaments, but the relevance of these findings for CBM complex assembly in cells remains unresolved. To uncouple cellular CARD11 recruitment of BCL10 and BCL10 filament assembly, we generated a BCL10-CARD11 fusion protein that links the C-terminus of BCL10 to the N-terminus of CARD11. When stably expressed in CARD11 KO Jurkat T cells, the BCL10-CARD11 fusion induced constitutive MALT1 activation. Furthermore, in CARD11 KO BJAB B cells, BCL10-CARD11 promoted constitutive NF-κB activation to a similar extent as CARD11 containing oncogenic driver mutations. Using structure-guided destructive mutations in the CARD11-BCL10 (CARD11 R35A) or BCL10-BCL10 (BCL10 R42E) interfaces, we demonstrate that chronic activation by the BCL10-CARD11 fusion protein was independent of the CARD11 CARD. However, activation strictly relied upon the ability of the BCL10 CARD to form oligomers. Thus, by combining distinct CARD mutations in the context of constitutively active BCL10-CARD11 fusion proteins, we provide evidence that BCL10-MALT1 recruitment to CARD11 and BCL10 oligomerization are interconnected processes, which bridge the CARD11 seed to downstream pathways in lymphocytes.

MALT1 activation by TRAF6 needs neither BCL10 nor CARD11.

The MALT1 (Mucosa associated lymphoid tissue lymphoma translocation protein 1) paracaspase couples antigen receptors on lymphocytes to downstream signaling events. Activation of MALT1 is known to involve stimulus-dependent CBM complex formation, that is, the recruitment of BCL10-bound MALT1 to a CARD-Coiled Coil protein. Beyond this canonical, CBM-dependent mechanism of MALT1 activation, recent studies suggest that MALT1 protease activity may be triggered by alternative mechanisms. For instance, the E3-ligase TRAF6 can activate MALT1 proteolytic function and induce MALT1 auto-cleavage. However, the interplay between CBM and TRAF6 with regard to MALT1 activation has remained incompletely elucidated. Here, by generating CRISPR/Cas9-derived knock-out Jurkat T-cells, we show that TRAF6 was dispensable for CARD11/BCL10-dependent MALT1 activation upon T-cell stimulation. However, ectopically-expressed TRAF6 could induce MALT1 activity in Jurkat T-cells devoid of either CARD11 or BCL10. These data provide unequivocal evidence that TRAF6-mediated MALT1 activation does not require the upstream scaffold CARD11 or the interaction between MALT1 and BCL10. Thus, TRAF6 may be part of a previously unidentified non-canonical pathway that triggers MALT1 protease activity independently of canonical CBM signalosomes.

Molecular architecture and regulation of BCL10-MALT1 filaments.

The CARD11-BCL10-MALT1 (CBM) complex triggers the adaptive immune response in lymphocytes and lymphoma cells. CARD11/CARMA1 acts as a molecular seed inducing BCL10 filaments, but the integration of MALT1 and the assembly of a functional CBM complex has remained elusive. Using cryo-EM we solved the helical structure of the BCL10-MALT1 filament. The structural model of the filament core solved at 4.9 Å resolution identified the interface between the N-terminal MALT1 DD and the BCL10 caspase recruitment domain. The C-terminal MALT1 Ig and paracaspase domains protrude from this core to orchestrate binding of mediators and substrates at the filament periphery. Mutagenesis studies support the importance of the identified BCL10-MALT1 interface for CBM complex assembly, MALT1 protease activation and NF-κB signaling in Jurkat and primary CD4 T-cells. Collectively, we present a model for the assembly and architecture of the CBM signaling complex and how it functions as a signaling hub in T-lymphocytes.

BCL10 - Bridging CARDs to Immune Activation.

Since the B-cell lymphoma/leukemia 10 (BCL10) protein was first described in 1999, numerous studies have elucidated its key functions in channeling adaptive and innate immune signaling downstream of CARMA/caspase-recruitment domain (CARD) scaffold proteins. While T and B cell antigen receptor (TCR/BCR) signaling induces the recruitment of BCL10 bound to mucosa-associated lymphoid tissue (MALT)1 to the lymphocyte-specific CARMA1/CARD11-BCL10-MALT1 (CBM-1) signalosome, alternative CBM complexes utilize different CARMA/CARD scaffolds in distinct innate or inflammatory pathways. BCL10 constitutes the smallest subunit in all CBM signalosomes, containing a 233 amino acid coding for N-terminal CARD as well as a C-terminal Ser/Thr-rich region. BCL10 forms filaments, thereby aggregating into higher-order clusters that mediate and amplify stimulation-induced signals, ultimately leading to MALT1 protease activation and canonical NF-κB and JNK signaling. BCL10 additionally undergoes extensive post-translational regulation involving phosphorylation, ubiquitination, MALT1-catalyzed cleavage, and degradation. Through these feedback and feed-forward events, BCL10 integrates positive and negative regulatory processes that govern the function as well as the dynamic assembly, disassembly, and destruction of CBM complexes. Thus, BCL10 is a critical regulator for activation as well as termination of immune cell signaling, revealing that its role extends far beyond that of a mere linking factor in CBM complexes.

Alternative splicing of MALT1 controls signalling and activation of CD4(+) T cells.

MALT1 channels proximal T-cell receptor (TCR) signalling to downstream signalling pathways. With MALT1A and MALT1B two conserved splice variants exist and we demonstrate here that MALT1 alternative splicing supports optimal T-cell activation. Inclusion of exon7 in MALT1A facilitates the recruitment of TRAF6, which augments MALT1 scaffolding function, but not protease activity. Naive CD4(+) T cells express almost exclusively MALT1B and MALT1A expression is induced by TCR stimulation. We identify hnRNP U as a suppressor of exon7 inclusion. Whereas selective depletion of MALT1A impairs T-cell signalling and activation, downregulation of hnRNP U enhances MALT1A expression and T-cell activation. Thus, TCR-induced alternative splicing augments MALT1 scaffolding to enhance downstream signalling and to promote optimal T-cell activation.