Norberto Gonzalez-Juarbe

Pore-Forming Toxins Induce Macrophage Necroptosis during Acute Bacterial Pneumonia.

Necroptosis is a highly pro-inflammatory mode of cell death regulated by RIP (or RIPK)1 and RIP3 kinases and mediated by the effector MLKL. We report that diverse bacterial pathogens that produce a pore-forming toxin (PFT) induce necroptosis of macrophages and this can be blocked for protection against Serratia marcescens hemorrhagic pneumonia. Following challenge with S. marcescens, Staphylococcus aureus, Streptococcus pneumoniae, Listeria monocytogenes, uropathogenic Escherichia coli (UPEC), and purified recombinant pneumolysin, macrophages pretreated with inhibitors of RIP1, RIP3, and MLKL were protected against death. Alveolar macrophages in MLKL KO mice were also protected during S. marcescens pneumonia. Inhibition of caspases had no impact on macrophage death and caspase-1 and -3/7 were determined to be inactive following challenge despite the detection of IL-1β in supernatants. Bone marrow-derived macrophages from RIP3 KO, but not caspase-1/11 KO or caspase-3 KO mice, were resistant to PFT-induced death. We explored the mechanisms for PFT-induced necroptosis and determined that loss of ion homeostasis at the plasma membrane, mitochondrial damage, ATP depletion, and the generation of reactive oxygen species were together responsible. Treatment of mice with necrostatin-5, an inhibitor of RIP1; GW806742X, an inhibitor of MLKL; and necrostatin-5 along with co-enzyme Q10 (N5/C10), which enhances ATP production; reduced the severity of S. marcescens pneumonia in a mouse intratracheal challenge model. N5/C10 protected alveolar macrophages, reduced bacterial burden, and lessened hemorrhage in the lungs. We conclude that necroptosis is the major cell death pathway evoked by PFTs in macrophages and the necroptosis pathway can be targeted for disease intervention.

Streptococcus pneumoniae Biofilm Formation Is Strain Dependent, Multifactorial, and Associated with Reduced Invasiveness and Immunoreactivity during Colonization

ABSTRACT Biofilms are thought to play an important role during colonization of the nasopharynx by Streptococcus pneumoniae, yet how they form in vivo and the determinants responsible remain unknown. Using scanning electron microscopy, we show that biofilm aggregates of increasing complexity form on murine nasal septa following intranasal inoculation. These biofilms were highly distinct from in vitro biofilms, as they were discontiguous and appeared to incorporate nonbacterial components such as intact host cells. Biofilms initially formed on the surface of ciliated epithelial cells and, as cells were sloughed off, were found on the basement membrane. The size and number of biofilm aggregates within nasal lavage fluid were digitally quantitated and revealed strain-specific capabilities that loosely correlated with the ability to form robust in vitro biofilms. We tested the ability of isogenic mutants deficient in CbpA, pneumolysin, hydrogen peroxide, LytA, LuxS, CiaR/H, and PsrP to form biofilms within the nasopharynx. This analysis revealed that CiaR/H was absolutely required for colonization, that PsrP and SpxB strongly impacted aggregate formation, and that other determinants affected aggregate morphology in a modest fashion. We determined that mice colonized with ΔpsrP mutants had greater levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-1β, and KC in nasal lavage fluid than did mice colonized with wild-type controls. This phenotype correlated with a diminished capacity of biofilm pneumococci to invade host cells in vitro despite enhanced attachment. Our results show that biofilms form during colonization and suggest that they may contribute to persistence through a hyperadhesive, noninvasive state that elicits a dampened cytokine response. IMPORTANCE This work demonstrates the first temporal characterization of Streptococcus pneumoniae biofilm formation in vivo. Our results show that the morphology of biofilms formed by both invasive and noninvasive clinical isolates in vivo is distinct from that of formed biofilms in vitro, yet propensity to form biofilms in vivo loosely correlates with the degree of in vitro biofilm formation on a microtiter plate. We show that host components, including intact host cells, influence the formation of in vivo structures. We also found that efficient biofilm formation in vivo requires multiple bacterial determinants. While some factors are essential for in vivo biofilm formation (CiaRH, PsrP, and SpxB), other factors are less critical (CbpA, LytA, LuxS, and pneumolysin). In comparison to their planktonic counterparts, biofilm pneumococci are hyperadhesive but less invasive and elicit a weaker proinflammatory cytokine response. These findings give insight into the requirements for and potential role of biofilms during prolonged asymptomatic colonization. This work demonstrates the first temporal characterization of Streptococcus pneumoniae biofilm formation in vivo. Our results show that the morphology of biofilms formed by both invasive and noninvasive clinical isolates in vivo is distinct from that of formed biofilms in vitro, yet propensity to form biofilms in vivo loosely correlates with the degree of in vitro biofilm formation on a microtiter plate. We show that host components, including intact host cells, influence the formation of in vivo structures. We also found that efficient biofilm formation in vivo requires multiple bacterial determinants. While some factors are essential for in vivo biofilm formation (CiaRH, PsrP, and SpxB), other factors are less critical (CbpA, LytA, LuxS, and pneumolysin). In comparison to their planktonic counterparts, biofilm pneumococci are hyperadhesive but less invasive and elicit a weaker proinflammatory cytokine response. These findings give insight into the requirements for and potential role of biofilms during prolonged asymptomatic colonization.

Severe Pneumococcal Pneumonia Causes Acute Cardiac Toxicity and Subsequent Cardiac Remodeling

Rationale: Up to one‐third of patients hospitalized with pneumococcal pneumonia experience major adverse cardiac events (MACE) during or after pneumonia. In mice, Streptococcus pneumoniae can invade the myocardium, induce cardiomyocyte death, and disrupt cardiac function following bacteremia, but it is unknown whether the same occurs in humans with severe pneumonia. Objectives: We sought to determine whether S. pneumoniae can (1) translocate the heart, (2) induce cardiomyocyte death, (3) cause MACE, and (4) induce cardiac scar formation after antibiotic treatment during severe pneumonia using a nonhuman primate (NHP) model. Methods: We examined cardiac tissue from six adult NHPs with severe pneumococcal pneumonia and three uninfected control animals. Three animals were rescued with antibiotics (convalescent animals). Electrocardiographic, echocardiographic, and serum biomarkers of cardiac damage were measured (troponin T, N‐terminal pro‐brain natriuretic peptide, and heart‐type fatty acid binding protein). Histological examination included hematoxylin and eosin staining, immunofluorescence, immunohistochemistry, picrosirius red staining, and transmission electron microscopy. Immunoblots were used to assess the underlying mechanisms. Measurements and Main Results: Nonspecific ischemic alterations were detected by electrocardiography and echocardiography. Serum levels of troponin T and heart‐type fatty acid binding protein were increased (P < 0.05) after pneumococcal infection in both acutely ill and convalescent NHPs. S. pneumoniae was detected in the myocardium of all NHPs with acute severe pneumonia. Necroptosis and apoptosis were detected in the myocardium of both acutely ill and convalescent NHPs. Evidence of cardiac scar formation was observed only in convalescent animals by transmission electron microscopy and picrosirius red staining. Conclusions: S. pneumoniae invades the myocardium and induces cardiac injury with necroptosis and apoptosis, followed by cardiac scarring after antibiotic therapy, in an NHP model of severe pneumonia.

Infiltrated Macrophages Die of Pneumolysin-Mediated Necroptosis following Pneumococcal Myocardial Invasion

ABSTRACT Streptococcus pneumoniae (the pneumococcus) is capable of invading the heart. Herein we observed that pneumococcal invasion of the myocardium occurred soon after development of bacteremia and was continuous thereafter. Using immunofluorescence microscopy (IFM), we observed that S. pneumoniae replication within the heart preceded visual signs of tissue damage in cardiac tissue sections stained with hematoxylin and eosin. Different S. pneumoniae strains caused distinct cardiac pathologies: strain TIGR4, a serotype 4 isolate, caused discrete pneumococcus-filled microscopic lesions (microlesions), whereas strain D39, a serotype 2 isolate, was, in most instances, detectable only using IFM and was associated with foci of cardiomyocyte hydropic degeneration and immune cell infiltration. Both strains efficiently invaded the myocardium, but cardiac damage was entirely dependent on the pore-forming toxin pneumolysin only for D39. Early microlesions caused by TIGR4 and microlesions formed by a TIGR4 pneumolysin-deficient mutant were infiltrated with CD11b+ and Ly6G-positive neutrophils and CD11b+ and F4/80-positive (F4/80+) macrophages. We subsequently demonstrated that macrophages in TIGR4-infected hearts died as a result of pneumolysin-induced necroptosis. The effector of necroptosis, phosphorylated mixed-lineage kinase domain-like protein (MLKL), was detected in CD11b+ and F4/80+ cells associated with microlesions. Likewise, treatment of infected mice and THP-1 macrophages in vitro with the receptor-interacting protein 1 kinase (RIP1) inhibitor necrostatin-5 promoted the formation of purulent microlesions and blocked cell death, respectively. We conclude that pneumococci that have invaded the myocardium are an important cause of cardiac damage, pneumolysin contributes to cardiac damage in a bacterial strain-specific manner, and pneumolysin kills infiltrated macrophages via necroptosis, which alters the immune response.

Requirement for Serratia marcescens cytolysin in a murine model of hemorrhagic pneumonia.

ABSTRACT Serratia marcescens, a member of the carbapenem-resistant Enterobacteriaceae, is an important emerging pathogen that causes a wide variety of nosocomial infections, spreads rapidly within hospitals, and has a systemic mortality rate of ≤41%. Despite multiple clinical descriptions of S. marcescens nosocomial pneumonia, little is known regarding the mechanisms of bacterial pathogenesis and the host immune response. To address this gap, we developed an oropharyngeal aspiration model of lethal and sublethal S. marcescens pneumonia in BALB/c mice and extensively characterized the latter. Lethal challenge (>4.0 × 106 CFU) was characterized by fulminate hemorrhagic pneumonia with rapid loss of lung function and death. Mice challenged with a sublethal dose (<2.0 × 106 CFU) rapidly lost weight, had diminished lung compliance, experienced lung hemorrhage, and responded to the infection with extensive neutrophil infiltration and histopathological changes in tissue architecture. Neutrophil extracellular trap formation and the expression of inflammatory cytokines occurred early after infection. Mice depleted of neutrophils were exquisitely susceptible to an otherwise nonlethal inoculum, thereby demonstrating the requirement for neutrophils in host protection. Mutation of the genes encoding the cytolysin ShlA and its transporter ShlB resulted in attenuated S. marcescens strains that failed to cause profound weight loss, extended illness, hemorrhage, and prolonged lung pathology in mice. This study describes a model of S. marcescens pneumonia that mimics known clinical features of human illness, identifies neutrophils and the toxin ShlA as a key factors important for defense and infection, respectively, and provides a solid foundation for future studies of novel therapeutics for this important opportunistic pathogen.

A non-human primate model of severe pneumococcal pneumonia

Rationale Streptococcus pneumoniae is the leading cause of community-acquired pneumonia and infectious death in adults worldwide. A non-human primate model is needed to study the molecular mechanisms that underlie the development of severe pneumonia, identify diagnostic tools, explore potential therapeutic targets, and test clinical interventions during pneumococcal pneumonia. Objective To develop a non-human primate model of pneumococcal pneumonia. Methods Seven adult baboons (Papio cynocephalus) were surgically tethered to a continuous monitoring system that recorded heart rate, temperature, and electrocardiography. Animals were inoculated with 109 colony-forming units of S. pneumoniae using bronchoscopy. Three baboons were rescued with intravenous ampicillin therapy. Pneumonia was diagnosed using lung ultrasonography and ex vivo confirmation by histopathology and immunodetection of pneumococcal capsule. Organ failure, using serum biomarkers and quantification of bacteremia, was assessed daily. Results Challenged animals developed signs and symptoms of pneumonia 4 days after infection. Infection was characterized by the presence of cough, tachypnea, dyspnea, tachycardia and fever. All animals developed leukocytosis and bacteremia 24 hours after infection. A severe inflammatory reaction was detected by elevation of serum cytokines, including Interleukin (IL)1Ra, IL-6, and IL-8, after infection. Lung ultrasonography precisely detected the lobes with pneumonia that were later confirmed by pathological analysis. Lung pathology positively correlated with disease severity. Antimicrobial therapy rapidly reversed symptomology and reduced serum cytokines. Conclusions We have developed a novel animal model for severe pneumococcal pneumonia that mimics the clinical presentation, inflammatory response, and infection kinetics seen in humans. This is a novel model to test vaccines and treatments, measure biomarkers to diagnose pneumonia, and predict outcomes.

Pore-forming toxin-mediated ion dysregulation leads to death receptor-independent necroptosis of lung epithelial cells during bacterial pneumonia

We report that pore-forming toxins (PFTs) induce respiratory epithelial cell necroptosis independently of death receptor signaling during bacterial pneumonia. Instead, necroptosis was activated as a result of ion dysregulation arising from membrane permeabilization. PFT-induced necroptosis required RIP1, RIP3 and MLKL, and could be induced in the absence or inhibition of TNFR1, TNFR2 and TLR4 signaling. We detected activated MLKL in the lungs from mice and nonhuman primates experiencing Serratia marcescens and Streptococcus pneumoniae pneumonia, respectively. We subsequently identified calcium influx and potassium efflux as the key initiating signals responsible for necroptosis; also that mitochondrial damage was not required for necroptosis activation but was exacerbated by MLKL activation. PFT-induced necroptosis in respiratory epithelial cells did not involve CamKII or reactive oxygen species. KO mice deficient in MLKL or RIP3 had increased survival and reduced pulmonary injury during S. marcescens pneumonia. Our results establish necroptosis as a major cell death pathway active during bacterial pneumonia and that necroptosis can occur without death receptor signaling.

Streptococcus pneumoniae in the heart subvert the host response through biofilm-mediated resident macrophage killing

For over 130 years, invasive pneumococcal disease has been associated with the presence of extracellular planktonic pneumococci, i.e. diplococci or short chains in affected tissues. Herein, we show that Streptococcus pneumoniae that invade the myocardium instead replicate within cellular vesicles and transition into non-purulent biofilms. Pneumococci within mature cardiac microlesions exhibited salient biofilm features including intrinsic resistance to antibiotic killing and the presence of an extracellular matrix. Dual RNA-seq and subsequent principal component analyses of heart- and blood-isolated pneumococci confirmed the biofilm phenotype in vivo and revealed stark anatomical site-specific differences in virulence gene expression; the latter having major implications on future vaccine antigen selection. Our RNA-seq approach also identified three genomic islands as exclusively expressed in vivo. Deletion of one such island, Region of Diversity 12, resulted in a biofilm-deficient and highly inflammogenic phenotype within the heart; indicating a possible link between the biofilm phenotype and a dampened host-response. We subsequently determined that biofilm pneumococci released greater amounts of the toxin pneumolysin than did planktonic or RD12 deficient pneumococci. This allowed heart-invaded wildtype pneumococci to kill resident cardiac macrophages and subsequently subvert cytokine/chemokine production and neutrophil infiltration into the myocardium. This is the first report for pneumococcal biofilm formation in an invasive disease setting. We show that biofilm pneumococci actively suppress the host response through pneumolysin-mediated immune cell killing. As such, our findings contradict the emerging notion that biofilm pneumococci are passively immunoquiescent.

Killing of Serratia marcescens biofilms with chloramphenicol.

Serratia marcescens is a Gram-negative bacterium with proven resistance to multiple antibiotics and causative of catheter-associated infections. Bacterial colonization of catheters mainly involves the formation of biofilm. The objectives of this study were to explore the susceptibility of S. marcescens biofilms to high doses of common antibiotics and non-antimicrobial agents. Biofilms formed by a clinical isolate of S. marcescens were treated with ceftriaxone, kanamycin, gentamicin, and chloramphenicol at doses corresponding to 10, 100 and 1000 times their planktonic minimum inhibitory concentration. In addition, biofilms were also treated with chemical compounds such as polysorbate-80 and ursolic acid. S. marcescens demonstrated susceptibility to ceftriaxone, kanamycin, gentamicin, and chloramphenicol in its planktonic form, however, only chloramphenicol reduced both biofilm biomass and biofilm viability. Polysorbate-80 and ursolic acid had minimal to no effect on either planktonic and biofilm grown S. marcescens. Our results suggest that supratherapeutic doses of chloramphenicol can be used effectively against established S. marcescens biofilms.

Bacterial Pore-Forming Toxins Promote the Activation of Caspases in Parallel to Necroptosis to Enhance Alarmin Release and Inflammation During Pneumonia

Pore-forming toxins are the most common virulence factor in pathogenic bacteria. They lead to membrane permeabilization and cell death. Herein, we show that respiratory epithelial cells (REC) undergoing bacterial pore-forming toxin (PFT)-induced necroptosis simultaneously experienced caspase activation independently of RIPK3. MLKL deficient REC treated with a pan-caspase inhibitor were protected in an additive manner against PFT-induced death. Subsequently, cleaved versions of caspases-2, -4 and -10 were detected within REC undergoing necroptosis by immunoblots and monoclonal antibody staining. Caspase activation was observed in lung samples from mice and non-human primates experiencing Gram-negative and Gram-positive bacterial pneumonia, respectively. During apoptosis, caspase activation normally leads to cell shrinkage, nuclear condensation, and immunoquiescent death. In contrast, caspase activity during PFT-induced necroptosis increased the release of alarmins to the extracellular milieu. Caspase-mediated alarmin release was found sufficient to activate resting macrophages, leading to Interleukin-6 production. In a mouse model of Gram-negative pneumonia, deletion of caspases -2 and -11, the mouse orthologue of caspase-4, reduced pulmonary inflammation, immune cell infiltration and lung damage. Thus, our study describes a previously unrecognized role for caspase activation in parallel to necroptosis, and indicates that their activity plays a critical pro-inflammatory role during bacterial pneumonia.