Biology subjects

Broz, P.

Publications and source records attributed to Broz, P..

13 recordsLinked to original sources

bioRxiv2026

Genome-wide CRISPRi maps pneumococcal attachment and intracellular survival during single infection and Influenza co-infection

Streptococcus pneumoniae remains a leading cause of bacterial pneumonia and frequently causes severe secondary infections following influenza A virus (IAV) infection. Yet, the genetic requirements underlying pneumococcal pathogenicity during viral co-infection remain incompletely understood. Here, we employed an inducible genome-wide CRISPR interference (CRISPRi) library to systematically investigate pneumococcal gene fitness during epithelial attachment and intracellular survival in macrophages under both single-infection and IAV co-infection conditions. Screening across multiple host cell types and viral strains identified 42 pneumococcal genes affecting host-cell attachment and 63 genes influencing intracellular survival. While global patterns of gene fitness were largely conserved between single infection and co-infection, genes involved in cell envelope biogenesis, including the undecaprenyl pyrophosphate phosphatase uppP, became increasingly important during co-infection. Pharmacological inhibition of cell wall biosynthesis recapitulated several genetic phenotypes and revealed enhanced sensitivity to bacitracin in virally altered host environments. Integration of CRISPRi screening with untargeted metabolomics uncovered extensive remodeling of nucleotide metabolism during infection. Disruption of nucleoside transport altered bacterial fitness, morphology, capsule expression, and host-cell attachment. Uridine availability emerged as a key regulator linking metabolic adaptation to virulence-associated traits, highlighting a trade-off between bacterial growth and adherence. Taken together, our study provides a genome-scale resource of pneumococcal fitness determinants during infection and demonstrates that influenza co-infection selectively reshapes bacterial genetic dependencies rather than globally altering pathogenicity programs. These findings identify cell envelope homeostasis and nucleotide metabolism as central regulators of pneumococcal virulence and reveal potential targets for future antimicrobial and anti-virulence strategies.

microbiology↗

bioRxiv2025

The unique lipopolysaccharide composition of Salmonella Typhimurium ST313 dampens pyroptosis and inflammasome activation and suppresses host cell death

In humans, non-typhoidal Salmonella (NTS) serovars, such as Salmonella enterica Typhimurium cause self-limiting gastroenteritis. However, invasive non-typhoidal Salmonella (iNTS) isolates ST313 have emerged in sub-Saharan Africa that cause a severe bacteremia in immunocompromised individuals including HIV-positive adults. Despite a decade of studies comparing ST313 to the classical diarrheal S. Typhimurium ST19 pathovariant, the molecular basis of the differences of pathogenesis between these bacteria remains unclear. Here we used primary human macrophages infected with the invasive S. Typhimurium ST313 D23580 isolate and the reference ST19 4/74 isolate as an in vitro model of infection. Transcriptomic profiling revealed that primary human macrophages infected with ST313 bacteria had distinct patterns of host cell death and inflammation responses compared to ST19-infected cells. Specifically, infection with ST313 isolate induced significantly less pyroptotic cell death which was associated with minimal processing of Gasdermin-D. We discovered that S. Typhimurium ST313 stimulated reduced IL-1{beta} release and a low-level activation of the upstream inflammasome signaling cascade with reduced Caspase-4 and Caspase-1 cleavage. To investigate the molecular basis of this phenotype we purified lipopolysaccharide (LPS) species from ST313 to identify physical properties and structures that were distinct from ST19 LPS. The ST313-derived LPS was less pro- inflammatory than LPS from ST19, mirroring the low inflammatory phenotype of the live bacteria. Together, our data reveal that the unique LPS of ST313 is responsible for the reduced level of pyroptosis in human macrophages. This phenotype is consistent with the intracellular persistence and dissemination of these pathogenic bacteria. AUTHOR SUMMARYNon-typhoidal Salmonella (NTS) is a foodborne microbial pathogen that causes a self- limiting gastroenteritis in humans. However, in recent decades invasive non-typhoidal Salmonella (iNTS) isolates have emerged in sub-Saharan Africa, and are currently causing severe bloodstream infections responsible for about 66,000 deaths each year, largely in immunocompromised individuals. To investigate differences in pathogenicity between iNTS and NTS strains, we studied the infection of primary human macrophage with an iNTS ST313 isolate from Malawi in comparison to a gastroenteritis-associated ST19 reference strain. Primary macrophages play a key role in clearing Salmonella infections by recognizing bacterial components such as lipopolysaccharide (LPS). LPS induces immune responses including activation of cytokine release, inflammation and host cell death for effective bacterial elimination. Our study revealed that the iNTS isolate survived better in human primary macrophages than the NTS strain due to reduced inflammatory cell death, weakened inflammasome activation and lower cytokine release. We suggest that the distinct LPS structure is responsible for these dampened immune responses which aid the spread and persistence of S. Typhimurium ST313 in the human host.

microbiology↗

bioRxiv2025

Sterile- or pathogen-induced endolysosomal damage activate the NLRP6 inflammasome in human intestinal epithelial cells

NLRP6 controls host defense against bacteria and viruses in the gastrointestinal tract by a poorly understood mechanism. Here, we report that NLRP6 forms an inflammasome upon endolysosomal damage caused by sterile triggers or bacterial pathogens such as Listeria monocytogenes in human intestinal epithelial cells (IECs). NLRP6 activation required Listeriolysin O-dependent cytosolic invasion of L. monocytogenes and drove IEC pyroptosis via ASC/Caspase-1-mediated GSDMD cleavage. NLRP6 inflammasome formation was independent of bacterial PAMPs, such as lipoteichoic acid or dsRNA, which were previously reported to activate NLRP6. L. monocytogenes mutants deficient in cell-to-cell spread or escape from secondary vacuoles induced less cell death, linking bacteria-induced endolysosomal damage to NLRP6 activation. Finally, sterile endolysosomal damage recapitulated pathogen-induced NLRP6 activation and drove IEC pyroptosis. In summary, our study reveals that NLRP6 allows IECs to detect endolysosomal damage, thereby not only making it responsive to pathogens but more generally to wide-ranging sources of pathological endolysosomal damage.

immunology↗

bioRxiv2024

NINJ1 is a restriction factor for HSV-1 in mouse macrophages

Restriction factors block multiple stages of viral infection. Here we describe how NINJ1 controls HSV-1 infection of mouse macrophages, a key cell type during systemic and local acute infection. We observed that Ninj1-/- macrophages are more susceptible to infection than WT cells. Ninj1-/- macrophages exhibited earlier and stronger expression of all kinetic classes of viral proteins, resulting in higher production of infectious viral particles, with initial differences seen in the first few hours post infection. Given the important role of NINJ1 during cell death, we next investigated if NINJ1 restricts HSV-1 in a manner related to its role in cell death. However, infected Ninj1-deficient cells did not exhibit differences in cell death compared to WT controls at early time points in infection where we observe a difference in infection rates. Instead, the higher infection rate in Ninj1-/- macrophages correlated to enhancement in the average numbers of viral particles entering each cell. Finally, we determined the consequences of the altered entry and found higher ISG RNA expression in infected Ninj1-/- cells, which we ascribe to both TLR signaling and STING-mediated recognition. Together, this indicates that the NINJ1 present on WT macrophages reduces HSV-1 entry, which has consequences for the inflammatory phenotype associated with HSV-1 infection.

microbiology↗

bioRxiv2024

Ninjurin-1 mediated plasma membrane rupture is a two-step process requiring cell swelling

Ninjurin-1 (NINJ1) executes cell lysis by polymerizing into filaments, yet how these form membrane lesions is unknown. Integrating cell-based assays with molecular dynamics simulations, we demonstrate that NINJ1-driven membrane rupture starts with the lateral association of NINJ1 dimers into double filaments. Subsequently, swelling of necrotic cells increases plasma membrane tension, leading to separation of the filament interface and NINJ1 lesion opening. Inhibiting cell swelling prevents this increase in tension, blocking NINJ1-mediated plasma membrane rupture without disrupting NINJ1 oligomerization, implicating membrane tension as key for opening NINJ1 lesions. The NINJ1 homolog NINJ2 oligomerizes but fails to form membrane lesions during cell death due to the greater stability of its dimer. Finally, using atomic force microscopy of NINJ1-containing proteoliposomes, we show that NINJ1 forms large pores by stabilizing membrane edges. In summary, our findings support a model whereby membrane tension drives a zipper-like opening of NINJ1 double filaments, initiating necrotic cell lysis.

immunology↗

bioRxiv2024

Cardiolipin Inhibits the Noncanonical Inflammasome by Preventing LPS Binding to Caspase 4/11 to Mitigate Endotoxemia in Vivo

In Gram-negative bacterial sepsis, excessive caspase 4/11 activation in response to circulating bacterial lipid LPS (endotoxemia) can cause organ damage and mortality. Current inhibitors of caspase 4/11 also block caspase 1 activity and are therefore not appealing clinical candidates for treating Gram-negative sepsis. Here, we identify double-unsaturated 18:2 cardiolipin as a selective inhibitor of caspase 4/11-dependent inflammatory cytokine secretion and pyroptosis, without affecting caspase-1 responses. Cardiolipin targets the CARD domain of caspase 4/11, impeding its interaction with LPS to restrain caspase 4/11 activation, thereby suppressing endotoxemia-induced systemic inflammation in vivo. Thus, we present cardiolipin as a promising candidate for preventing endotoxemia- induced sequelae in sepsis while preserving caspase-1-driven anti-microbial immune responses. By identifying cardiolipin as a specific caspase 4/11 inhibitor, we provide an urgently-needed tool for studying caspase 4/11 functions in inflammatory pathways, and open the way to studies of noncanonical inflammasome regulation by endogenous cardiolipin.

immunology↗

bioRxiv2022

Gasdermin D is the only Gasdermin that provides non-redundant protection against acute Salmonella gut infection

Gasdermins (GSDMs) share a common functional domain structure and are best known for their capacity to form membrane pores. These pores are hallmarks of a specific form of cell death called pyroptosis and mediate the secretion of pro-inflammatory cytokines such as interleukin 1{beta} (IL1{beta}) and interleukin 18 (IL18). Thereby, Gasdermins have been implicated in various immune responses against cancer and infectious diseases such as acute Salmonella Typhimurium (S.Tm) gut infection. However, to date, we lack a comprehensive functional assessment of the different Gasdermins (GSDMA-E) during S.Tm infection in vivo. Here, we have performed littermate-controlled oral S.Tm infections to investigate the impact of all murine Gasdermins. While GSDMA, -C and -E appear dispensable, we show that GSDMD (i) restricts S.Tm loads in the gut tissue and systemic organs, (ii) controls gut inflammation kinetics, and (iii) prevents epithelium disruption by 72h of the infection. Full protection requires GSDMD expression by both bone-marrow-derived lamina propria cells and intestinal epithelial cells (IECs). In vivo experiments, 3D- and 2D-enteroid infections further show that infected IEC extrusion proceeds also without GSDMD, but that GSDMD controls the permeabilization and morphology of the extruding cells and affects extrusion kinetics. As such, this work identifies a non-redundant multipronged role of GSDMD in mucosal tissue defence against a common enteric pathogen. HIGHLIGHTSO_LIGasdermin D restricts Salmonella Typhimurium (S.Tm) translocation across the gut tissue, controls gut inflammation kinetics, and prevents epithelium disruption by 72h of the infection. C_LIO_LIGasdermins A, C and E appear dispensable for protection against acute S.Tm gut infection. C_LIO_LIGasdermin D in bone-marrow-derived lamina propria cells and intestinal epithelial cells complement each other to suppress gut tissue S.Tm loads. C_LIO_LIGasdermin D is not required for extrusion of infected intestinal epithelial cells but drives their permeabilization and affects qualitative features of the extrusion process. C_LI

immunology↗

bioRxiv2022

The Opto-inflammasome in zebrafish: a tool to study cell and tissue responses to speck formation and cell death

The inflammasome is a conserved system for the intracellular detection of danger or pathogen signals. By forming a large intracellular multiprotein signaling platform, it activates downstream effectors that initiate a rapid necrotic programmed cell death (PCD) termed pyroptosis and activation and secretion of pro-inflammatory cytokines to warn and activate surrounding cells. However, inflammasome activation is difficult to control on a single-cell level using canonical triggers. We constructed Opto-Asc, a light-responsive form of the inflammasome adaptor protein ASC (Apoptosis-Associated Speck-Like Protein Containing a CARD) which allows tight control of inflammasome formation in vivo. We introduced a cassette of this construct under the control of a heat shock element into zebrafish in which we can now induce Asc inflammasome (speck) formation in single cells of the skin. We find that cell death resulting from Asc speck formation is morphological distinct from apoptosis in periderm but not in basal cells. Asc induced PCD in can lead to apical or basal extrusion from the periderm. The apical extrusion in periderm cells depends on Caspb but and triggers a strong Ca2+ signaling response in nearby cells.

immunology↗

bioRxiv2021

Optogenetic activators of apoptosis, necroptosis and pyroptosis for probing cell death dynamics and bystander cell responses

Targeted and specific induction of cell death in individual or groups of cells holds the potential for new insights into the response of tissues or organisms to different forms of death. Here we report the development of optogenetically-controlled cell death effectors (optoCDEs), a novel class of optogenetic tools that enables light-mediated induction of three types of programmed cell death (PCD) - apoptosis, pyroptosis and necroptosis - using Arabidopsis thaliana photosensitive protein Cryptochrome2. OptoCDEs enable rapid and highly specific induction of PCD in human, mouse and zebrafish cells and are suitable for a wide range of applications, such as sub-lethal cell death induction or precise elimination of single cells or cell populations in vitro and in vivo. As the proof-of-concept, we utilize optoCDEs to assess the differences in the neighboring cell response to apoptotic or necrotic PCD, revealing a new role for shingosine-1-phosphate signaling in regulating the efferocytosis of apoptotic cell by epithelia.

cell biology↗

bioRxiv2021

The Card19 locus of murine chromosome 13 regulates terminal cell lysis downstream of caspase activation and Gasdermin-D cleavage

Cell death plays a critical role in inflammatory responses. During pyroptosis, inflammatory caspases cleave Gasdermin D (GSDMD) to release an N-terminal fragment that generates plasma membrane pores that mediate cell lysis and IL-1 cytokine release. Terminal cell lysis and IL-1{beta} release following caspase activation can be uncoupled in certain cell types or in response to particular stimuli, a state termed hyperactivation. However, the factors and mechanisms that regulate terminal cell lysis downstream of GSDMD cleavage remain poorly understood. In the course of studies to define regulation of pyroptosis during Yersinia infection, we identified a line of Card19-deficient mice (Card19lxcn) whose macrophages were protected from cell lysis and showed reduced apoptosis and pyroptosis, yet had wild-type levels of caspase activation, IL-1 secretion, and GSDMD cleavage. Unexpectedly, CARD19, a mitochondrial CARD-containing protein, was not directly responsible for this, as two independently-generated CRISPR/Cas9 Card19 knockout mice showed no defect in macrophage cell lysis, and expression of CARD19 in Card19lxcn macrophages did not restore cell lysis. Card19 is located on chromosome 13, adjacent to Ninj1, which was recently reported to regulate cell lysis downstream of GSDMD activation. Intriguingly, RNA-seq and western blotting revealed that Card19lxcn BMDMs are hypomorphic for NINJ1 expression, and reconstitution of Ninj1 in Card19lxcn immortalized BMDMs restored cell lysis. Card19lxcn mice exhibited significantly increased susceptibility to Yersinia infection, demonstrating that cell lysis itself plays a key role in protection against bacterial infection. Our findings identify genetic targeting of Card19 being responsible for off-target effects on the adjacent Ninj1 gene, thereby disrupting the ability of macrophages to undergo plasma membrane rupture downstream of gasdermin cleavage and impacting host survival and bacterial control during Yersinia infection. Author SummaryProgrammed cell death is critical for regulating tissue homeostasis and host defense against infection. Pyroptosis is an inflammatory form of programmed cell death that couples cell lysis with release of inflammatory cytokines. Cell lysis is triggered by activation of particular intracellular pore forming proteins, but how regulation of cell lysis occurs is not well understood. Genetic targeting of Card19 on chromosome 13 resulted in decreased expression of the adjacent gene, Ninj1 which was recently found to regulate terminal lysis events in response to cell death-inducing stimuli. We found that macrophages from Card19-deficient mice were resistant to multiple forms of cell death in response to a variety of inflammatory stimuli, including canonical and non-canonical inflammasome activation, as well as triggers of cell-extrinsic apoptosis. Notably, Card19-deficient mice were more susceptible to Yersinia infection, indicating that cell lysis contributes to control of bacterial infections. Our data provide new insight into the impact of terminal cell lysis on control of bacterial infection and highlight the role of additional factors that regulate lytic cell death downstream of gasdermin cleavage.

immunology↗

bioRxiv2021

RIPK1 activates distinct gasdermins in macrophages and neutrophils upon pathogen blockade of innate immune signalling

Injection of effector proteins to block host innate immune signalling is a common strategy used by many pathogenic organisms to establish an infection. Pathogenic Yersinia species for example inject the acetyltransferase YopJ into target cells to inhibit NF-{kappa}B and MAPK signalling. To counteract this, detection of YopJ activity in myeloid cells promotes the assembly of a RIPK1-caspase-8 death-inducing platform that confers antibacterial defence. While recent studies revealed that caspase-8 cleaves the pore-forming protein, gasdermin D (GSDMD) to trigger pyroptosis in macrophages, whether RIPK1 activates additional substrates downstream of caspase-8 to promote host defence is unclear. Here, we report that the related gasdermin family member gasdermin E (GSDME) is activated upon detection of YopJ activity in a RIPK1 kinase-dependent manner. Specifically, GSDME promotes neutrophil pyroptosis and IL-1{beta} release, which is critical for anti-Yersinia defence. During in vivo infection, IL-1{beta} neutralisation increases bacterial burden in wild type but not Gsdme-deficient mice. Thus, our study establishes GSDME as an important mediator that counteracts pathogen blockade of innate immune signalling.

immunology↗

bioRxiv2019

Type-3 Secretion System-induced pyroptosis protects Pseudomonas against cell-autonomous immunity

Inflammasome-induced pyroptosis comprises a key cell-autonomous immune process against intracellular bacteria, namely the generation of dying cell structures. These so-called pore-induced intracellular traps (PITs) entrap and weaken intracellular microbes. However, the immune importance of pyroptosis against extracellular pathogens remains unclear. Here, we report that Type-3 secretion system (T3SS)-expressing Pseudomonas aeruginosa (P. aeruginosa) escaped PIT immunity by inducing a NLRC4 inflammasome-dependent macrophage pyroptosis response in the extracellular environment. To the contrary, phagocytosis of Salmonella Typhimurium promoted NLRC4-dependent PIT formation and the subsequent bacterial caging. Remarkably, T3SS-deficient Pseudomonas were efficiently sequestered within PIT-dependent caging, which favored exposure to neutrophils. Conversely, both NLRC4 and caspase-11 deficient mice presented increased susceptibility to T3SS-deficient P. aeruginosa challenge, but not to T3SS-expressing P. aeruginosa. Overall, our results uncovered that P. aeruginosa uses its T3SS to overcome inflammasome-triggered pyroptosis, which is primarily effective against intracellular invaders.\n\nImportanceAlthough innate immune components confer host protection against infections, the opportunistic bacterial pathogen Pseudomonas aeruginosa (P. aeruginosa) exploits the inflammatory reaction to thrive. Specifically the NLRC4 inflammasome, a crucial immune complex, triggers an Interleukin (IL)-1{beta} and -18 deleterious host response to P. aeruginosa. Here, we provide evidence that, in addition to IL-1 cytokines, P. aeruginosa also exploits the NLRC4 inflammasome-induced pro-inflammatory cell death, namely pyroptosis, to avoid efficient uptake and killing by macrophages. Therefore, our study reveals that pyroptosis-driven immune effectiveness mainly depends on P. aeruginosa localization. This paves the way toward our comprehension of the mechanistic requirements for pyroptosis effectiveness upon microbial infections and may initiate targeted approaches in order to ameliorate the innate immune functions to infections.\n\nGraphical abstractMacrophages infected with T3SS-expressing P. aeruginosa die in a NLRC4-dependent manner, which allows bacterial escape from PIT-mediated cell-autonomous immunity and neutrophil efferocytosis. However, T3SS-deficient P. aeruginosa is detected by both NLRC4 and caspase-11 inflammasomes, which promotes bacterial trapping and subsequent efferocytosis of P. aeruginosa-containing-PITs by neutrophils.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC=\"FIGDIR/small/650333v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (15K):\norg.highwire.dtl.DTLVardef@17e05c4org.highwire.dtl.DTLVardef@aca55forg.highwire.dtl.DTLVardef@9d2853org.highwire.dtl.DTLVardef@1c275f9_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2017

Insights into Gasdermin D activation from the crystal structure of its C-terminal domain

Gasdermin D (GSDMD) is the central executioner of pyroptosis, a proinflammatory type of cell death. GSDMD is activated by the proinflammatory caspase-1 and caspase-11 via proteolytic cleavage in the linker connecting its N-terminal and C-terminal domain (GSDMDNterm, Cterm). The released N-terminal domain is sufficient to form pores in the plasma membrane, resulting in swelling and subsequent rupture of the cell. Here, we report the crystal structure of the autoinhibitory C-terminal domain of GSDMD at 2.04 [A] resolution to further analyse determinants of GSDMD activation. GSDMDCterm adopts a compact helical fold unique to gasdermin proteins. Structural analysis and comparison to other gasdermin proteins reveals a conserved key interface for interactions between GSDMDNterm and GSDMDCterm. Variations in two additional surface patches involved in interdomain interactions in full-length gasdermins suggest a role of these regions in modulating activation pathways, in agreement with biochemical characterization of different gasdermins.

molecular biology↗