Murine Pneumonia Model Research Articles

Polyclonal anti-whole cell IgY passive immunotherapy shields against P. aeruginosa-induced acute pneumonia and burn wound infections in murine models

Pseudomonas aeruginosa (PA) is a multidrug-resistant (MDR) opportunistic pathogen causing severe hospital-, and community-acquired infections worldwide. Thus, the development of effective immunotherapy-based treatments is essential to combat the MDR-PA infections. In the current study, we evaluated the protective efficacy of polyclonal avian antibodies raised against inactivated whole cells of the PAO1 strain in murine models of acute pneumonia and burn wound. The efficacy of generated antibodies was evaluated against different PA strains through several in vitro, ex vivo and in vivo experiments. The results showed that the anti-PAO1-IgY effectively reduced the motility, biofilm formation and cell internalization ability, and enhanced the opsonophagocytic killing of PA strains through the formation of immobilized bacteria and induction of increased cell surface hydrophobicity. Furthermore, immunotherapy with anti-PAO1-IgY completely protected mice against all PA strains in both acute pneumonia and burn wound murine models. It was found to reduce the bacterial loads in infected burned mice through interfering with virulence factors that play vital roles in the early stages of PA infection, such as colonization and cell internalization. The immunotherapy with anti-PAO1-IgYs could be instrumental in developing effective therapies aimed at reducing the morbidity and mortality associated with PA infections.

Azithromycin regulates bacterial virulence and immune response in a murine model of ceftazidime-treated Pseudomonas aeruginosa acute pneumonia.

Pseudomonas aeruginosa (PA) remains one of the leading causes of nosocomial acute pneumonia. The array of virulence factors expressed by PA and the intense immune response associated with PA pneumonia play a major role in the severity of these infections. New therapeutic approaches are needed to overcome the high resistance of PA to antibiotics and to reduce the direct damage to host tissues. Through its immunomodulatory and anti-virulence effects, azithromycin (AZM) has demonstrated clinical benefits in patients with chronic PA respiratory infections. However, there is relatively little evidence in PA acute pneumonia. We investigated the effects of AZM, as an adjunctive therapy combined with ceftazidime (CAZ), in a murine model of PA acute pneumonia. We observed that the combined therapy (i) reduces the weight loss of mice 24 h post-infection (hpi), (ii) decreases neutrophil influx into the lungs at 6 and 24 hpi, while this effect is absent in a LPS-induced pneumonia or when PA is pretreated with antibiotics and mice do not receive any antibiotics, and that (iii) AZM, alone or with CAZ, modulates the expression of PA quorum sensing regulators and virulence factors (LasI, LasA, PqsE, PhzM, ExoS). Our findings support beneficial effects of AZM with CAZ on PA acute pneumonia by both bacterial virulence and immune response modulations. Further investigations are needed to clarify the exact underlying mechanisms responsible for the reduction of the neutrophils influx and to better discriminate between direct immunomodulatory properties of AZM, and indirect effects on neutrophilia resulting from bacterial virulence modulation.

Perillaldehyde: A promising antibacterial agent for the treatment of pneumonia caused by Acinetobacter baumannii infection

Perillaldehyde is a monoterpene compound mainly from the medicinal plant Perilla frutescens (L.) Britt., which has hypolipidemic, antioxidant, antibacterial and anti-inflammatory functions. In this investigation, we discovered that Perillaldehyde had powerful antimicrobial activity against Acinetobacter baumannii 5F1, and its minimum inhibitory concentration was 287.08 μg/mL. A. baumannii is a conditionally pathogenic bacterium with a high clinical resistance rate and is a major source of hospital infections, especially in intensive care units, which is one of the main causes of pneumonia. Inflammatory immune response is characteristic of pneumonia caused by A. baumannii infection. The results of our in vitro experiments indicate that Perillaldehyde disrupts the cell membrane of A. baumannii 5F1 and inhibits its quorum sensing to inhibit biofilm formation, among other effects. With an experimental model of murine pneumonia, we investigated that Perillaldehyde decreased NLRP3 inflammasome activation and TNF-α expression in lung tissues by inhibiting the NF-κB pathway, and also impacted MAPKs protein signaling pathway through the activation of TLR4. Notably, the use of high doses of Perillaldehyde for the treatment of pneumonia caused by A. baumannii 5F1 infection resulted in a survival rate of up to 80 % in mice. In summary, we demonstrated that Perillaldehyde is promising as a new drug for the treatment of pneumonia caused by A. baumannii 5F1 infection.

The Group A Streptococcal Vaccine Candidate VAX-A1 Protects against Group B Streptococcus Infection via Cross-Reactive IgG Targeting Virulence Factor C5a Peptidase.

Group B Streptococcus (Streptococcus agalactiae or GBS) is the leading infectious cause of neonatal mortality, causing roughly 150,000 infant deaths and stillbirths annually across the globe. Approximately 20% of pregnant women are asymptomatically colonized by GBS, which is a major risk factor for severe fetal and neonatal infections as well as preterm birth, low birth weight, and neurodevelopmental abnormalities. Current clinical interventions for GBS infection are limited to antibiotics, and no vaccine is available. We previously described VAX-A1 as a highly effective conjugate vaccine against group A Streptococcus that is formulated with three antigens, SpyAD, streptolysin O, and C5a peptidase (ScpA). ScpA is a surface-expressed, well-characterized GAS virulence factor that shares nearly identical sequences with the lesser studied GBS homolog ScpB. Here, we show that GBS C5a peptidase ScpB cleaves human complement factor C5a and contributes to disease severity in the murine models of pneumonia and sepsis. Furthermore, antibodies elicited by GAS C5a peptidase bind to GBS in an ScpB-dependent manner, and VAX-A1 immunization protects mice against lethal GBS heterologous challenge. These findings support the contribution of ScpB to GBS virulence and underscore the importance of choosing vaccine antigens; a universal GAS vaccine such as VAX-A1 whose formulation includes GAS C5a peptidase may have additional benefits through some measure of cross-protection against GBS infections.

Klebsiella pneumoniae clinical isolates with features of both multidrug-resistance and hypervirulence have unexpectedly low virulence

Klebsiella pneumoniae has been classified into two types, classical K. pneumoniae (cKP) and hypervirulent K. pneumoniae (hvKP). cKP isolates are highly diverse and important causes of nosocomial infections; they include globally disseminated antibiotic-resistant clones. hvKP isolates are sensitive to most antibiotics but are highly virulent, causing community-acquired infections in healthy individuals. The virulence phenotype of hvKP is associated with pathogenicity loci responsible for siderophore and hypermucoid capsule production. Recently, convergent strains of K. pneumoniae, which possess features of both cKP and hvKP, have emerged and are cause of much concern. Here, we screen the genomes of 2,608 multidrug-resistant K. pneumoniae isolates from the United States and identify 47 convergent isolates. We perform phenotypic and genomic characterization of 12 representative isolates. These 12 convergent isolates contain a variety of antimicrobial resistance plasmids and virulence plasmids. Most convergent isolates contain aerobactin biosynthesis genes and produce more siderophores than cKP isolates but not more capsule. Unexpectedly, only 1 of the 12 tested convergent isolates has a level of virulence consistent with hvKP isolates in a murine pneumonia model. These findings suggest that additional studies should be performed to clarify whether convergent strains are indeed more virulent than cKP in mouse and human infections.

Efficacy of a Pseudomonas aeruginosa serogroup O9 vaccine.

There are currently no approved vaccines against the opportunistic pathogen Pseudomonas aeruginosa. Among vaccine targets, the lipopolysaccharide (LPS) O antigen of P. aeruginosa is the most immunodominant protective candidate. There are 20 different O antigens composed of different repeat sugar structures conferring serogroup specificity, and 10 are found most frequently in infection. Thus, one approach to combat infection by P. aeruginosa could be to generate immunity with a vaccine cocktail that includes all these serogroups. Serogroup O9 is 1 of the 10 serogroups commonly found in infection, but it has never been developed into a vaccine, due in part to the acid-labile nature of the O9 polysaccharide. Our laboratory has previously shown that intranasal administration of an attenuated Salmonella strain expressing the P. aeruginosa serogroup O11 LPS O antigen was effective in clearing bacteria and preventing mortality in mice following intranasal challenge with serogroup O11 P. aeruginosa. Consequently, we set out to develop a P. aeruginosa serogroup O9 vaccine using a similar approach. Here, we show that Salmonella expressing serogroup O9 triggered an antibody-mediated immune response following intranasal administration to mice and that it conferred protection from P. aeruginosa serogroup O9 in a murine model of acute pneumonia.

Bacterial outer membrane vesicles bound to bacteriophages modulate neutrophil responses to bacterial infection.

Pseudomonas aeruginosa is a major human pathogen, particularly effective at colonizing the airways of patients with cystic fibrosis. Bacteriophages are highly abundant at infection sites, but their impact on mammalian immunity remains unclear. We previously showed that Pf4, a temperate filamentous bacteriophage produced by P. aeruginosa, modifies the innate immune response to P. aeruginosa infections via TLR3 signaling, but the underlying mechanisms remained unclear. Notably, Pf4 is a single-stranded DNA and lysogenic phage, and its production does not typically result in lysis of its bacterial host. We identified previously that internalization of Pf4 by human or murine immune cells triggers maladaptive viral pattern recognition receptors and resulted in bacterial persistence based on the presence of phage RNA. We report now that Pf4 phage dampens inflammatory responses to bacterial endotoxin and that this is mediated in part via bacterial vesicles attached to phage particles. Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and play a key role in host pathogen interaction. Recently, evidence has emerged that OMVs differentially package small RNAs. In this study, we show that Pf4 are decorated with OMVs that remain affixed to Pf4 despite of purification steps. These phages are endocytosed by human cells and delivered to endosomal vesicles. We demonstrate that short RNAs within the OMVs form hairpin structures that trigger TLR3-dependent type I interferon production and antagonize production of antibacterial cytokines and chemokines. In particular, Pf4 phages inhibit CXCL5, preventing efficient neutrophil chemotaxis in response to endotoxin. Moreover, blocking IFNAR or TLR3 signaling abrogates the effect of Pf4 bound to OMVs on macrophage activation. In a murine acute pneumonia model, mice treated with Pf4 associated with OMVs show significantly less neutrophil infiltration in BAL fluid than mice treated with purified Pf4. These changes in macrophage phenotype are functionally relevant: conditioned media from cells exposed to Pf4 decorated with OMVs are significantly less effective at inducing neutrophil migration in vitro and in vivo. These results suggest that Pf4 phages alter innate immunity to bacterial endotoxin and OMVs, potentially dampening inflammation at sites of bacterial colonization or infection.

Echinacoside, a promising sortase A inhibitor, combined with vancomycin against murine models of MRSA-induced pneumonia.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium responsible for a range of severe infections, such as skin infections, bacteremia, and pneumonia. Due to its antibiotic-resistant nature, current research focuses on targeting its virulence factors. Sortase A (SrtA) is a transpeptidase that anchors surface proteins to the bacterial cell wall and is involved in adhesion and invasion to host cells. Through fluorescence resonance energy transfer (FRET), we identified echinacoside (ECH), a natural polyphenol, as a potential SrtA inhibitor with an IC50 of 38.42μM in vitro. It was demonstrated that ECH inhibited SrtA-mediated S. aureus fibrinogen binding, surface protein A anchoring, and biofilm formation. The fluorescence quenching assay determined the binding mode of ECH to SrtA and calculated the KA-binding constant of 3.09 × 105L/mol, demonstrating the direct interaction between the two molecules. Molecular dynamics simulations revealed that ECH-SrtA interactions occurred primarily at the binding sites of A92G, A104G, V168A, G192A, and R197A. Importantly, the combination of ECH and vancomycin offered protection against murine models of MRSA-induced pneumonia. Therefore, ECH may serve as a potential antivirulence agent against S. aureus infections, either alone or in combination with vancomycin.

Ketogenesis promotes tolerance to Pseudomonas aeruginosa pulmonary infection

Pseudomonas aeruginosa is a common cause of pulmonary infection. As a Gram-negative pathogen, it can initiate a brisk and highly destructive inflammatory response; however, most hosts become tolerant to the bacterial burden, developing chronic infection. Using a murine model of pneumonia, we demonstrate that this shift from inflammation to disease tolerance is promoted by ketogenesis. In response to pulmonary infection, ketone bodies are generated in the liver and circulate to the lungs where they impose selection for P.aeruginosa strains unable to display surface lipopolysaccharide (LPS). Such keto-adapted LPS strains fail to activate glycolysis and tissue-damaging cytokines and, instead, facilitate mitochondrial catabolism of fats and oxidative phosphorylation (OXPHOS), which maintains airway homeostasis. Within the lung, P.aeruginosa exploits the host immunometabolite itaconate to further stimulate ketogenesis. This environment enables host-P.aeruginosa coexistence, supporting both pathoadaptive changes in the bacteria and the maintenance of respiratory integrity via OXPHOS.

Metallo-sideromycin as a dual functional complex for combating antimicrobial resistance

The rapid emergence of antimicrobial resistance (AMR) pathogens highlights the urgent need to approach this global burden with alternative strategies. Cefiderocol (Fetroja®) is a clinically-used sideromycin, that is utilized for the treatment of severe drug-resistant infections, caused by Gram-negative bacteria; there is evidence of cefiderocol-resistance occurring in bacterial strains however. To increase the efficacy and extend the life-span of sideromycins, we demonstrate strong synergisms between cefiderocol and metallodrugs (e.g., colloidal bismuth citrate (CBS)), against Pseudomonas aeruginosa and Burkholderia cepacia. Moreover, CBS enhances cefiderocol efficacy against biofilm formation, suppresses the resistance development in P. aeruginosa and resensitizes clinically isolated resistant P. aeruginosa to cefiderocol. Notably, the co-therapy of CBS and cefiderocol significantly increases the survival rate of mice and decreases bacterial loads in the lung in a murine acute pneumonia model. The observed phenomena are partially attributable to the competitive binding of Bi3+ to cefiderocol with Fe3+, leading to enhanced uptake of Bi3+ and reduced levels of Fe3+ in cells. Our studies provide insight into the antimicrobial potential of metallo-sideromycins.

Pseudomonas aeruginosa pulmonary infection results in S100A8/A9-dependent cardiac dysfunction.

Pseudomonas aeruginosa (P.a.) infection accounts for nearly 20% of all cases of hospital acquired pneumonia with mortality rates >30%. P.a. infection induces a robust inflammatory response, which ideally enhances bacterial clearance. Unfortunately, excessive inflammation can also have negative effects, and often leads to cardiac dysfunction with associated morbidity and mortality. However, it remains unclear how P.a. lung infection causes cardiac dysfunction. Using a murine pneumonia model, we found that P.a. infection of the lungs led to severe cardiac left ventricular dysfunction and electrical abnormalities. More specifically, we found that neutrophil recruitment and release of S100A8/A9 in the lungs activates the TLR4/RAGE signaling pathways, which in turn enhance systemic inflammation and subsequent cardiac dysfunction. Paradoxically, global deletion of S100A8/A9 did not improve but aggravated cardiac dysfunction and mortality likely due to uncontrolled bacterial burden in the lungs and heart. Our results indicate that P.a. infection induced release of S100A8/9 is double-edged, providing increased risk for cardiac dysfunction yet limiting P.a. growth.

Staphylococcus aureus SigS Induces Expression of a Regulatory Protein Pair That Modulates Its mRNA Stability.

SigS is the sole extracytoplasmic function sigma factor in Staphylococcus aureus and is necessary for virulence, immune evasion, and adaptation to toxic chemicals and environmental stressors. Despite the contribution of SigS to a myriad of critical phenotypes, the downstream effectors of SigS-dependent pathogenesis, immune evasion, and stress adaptation remain elusive. To address this knowledge gap, we analyzed the S. aureus transcriptome following transient overexpression of SigS. We identified a bicistronic transcript, upregulated 1,000-fold, containing two midsized genes, each containing single domains of unknown function (DUFs). We renamed these genes SigS-regulated orfA (sroA) and SigS-regulated orfB (sroB). We demonstrated that SigS regulation of the sroAB operon is direct by using in vitro transcription analysis. Using Northern blot analysis, we also demonstrated that SroA and SroB have opposing autoregulatory functions on the transcriptional architecture of the sigS locus, with SroA stimulating SigS mRNA levels and SroB stimulating s750 (SigS antisense) levels. We hypothesized that these opposing regulatory effects were due to a direct interaction. We subsequently demonstrated a direct interaction between SroA and SroB using an in vivo surrogate genetics approach via bacterial adenylate cyclase-based two-hybrid (BACTH) analysis. We demonstrated that the SroA effect on SigS is at the posttranscriptional level of mRNA stability, highlighting a mechanism likely used by S. aureus to tightly control SigS levels. Finally, we demonstrate that the sroAB locus promotes virulence in a murine pneumonia model of infection. IMPORTANCE SigS is necessary for S. aureus virulence, immune evasion, and adaptation to chemical and environmental stressors. These processes are critically important for the ability of S. aureus to cause disease. However, the SigS-dependent transcriptome has not been identified, hindering our ability to identify downstream effectors of SigS that contribute to these pathogenic and adaptive phenotypes. Here, we identify a regulatory protein pair that is a major direct target of SigS, known as SroA and SroB. SroA also acts to stimulate SigS expression at the posttranscriptional level of RNA turnover, providing insight into intrinsically low levels of SigS. The discovery of SroA and SroB increases our understanding of SigS and the S. aureus pathogenesis process.

Immunostimulatory DNA Hydrogel Enhances Protective Efficacy of Nanotoxoids against Bacterial Infection.

While vaccines have been highly successful in protecting against various infections, there are still many high-priority pathogens for which there are no clinically approved formulations. To overcome this challenge, researchers have explored the use of nanoparticulate strategies for more effective antigen delivery to the immune system. Along these lines, nanotoxoids are a promising biomimetic platform that leverages cell membrane coating technology to safely deliver otherwise toxic bacterial antigens in their native form for antivirulence vaccination. Here, in order to further boost their immunogenicity, nanotoxoids formulated against staphylococcal α-hemolysin are embedded into a DNA-based hydrogel with immunostimulatory CpG motifs. The resulting nanoparticle-hydrogel composite is injectable and improves the in vivo delivery of vaccine antigens while simultaneously stimulating nearby immune cells. This leads to elevated antibody production and stronger antigen-specific cellular immune responses. In murine models of pneumonia and skin infection caused by methicillin-resistant Staphylococcus aureus, mice vaccinated with the hybrid vaccine formulation are well-protected. This work highlights the benefits of combining nanoparticulate antigen delivery systems with immunostimulatory hydrogels into a single platform, and the approach can be readily generalized to a wide range of infectious diseases.

Replicative Acinetobacter baumannii strains interfere with phagosomal maturation by modulating the vacuolar pH.

Bacterial pneumonia is a common infection of the lower respiratory tract that can afflict patients of all ages. Multidrug-resistant strains of Acinetobacter baumannii are increasingly responsible for causing nosocomial pneumonias, thus posing an urgent threat. Alveolar macrophages play a critical role in overcoming respiratory infections caused by this pathogen. Recently, we and others have shown that new clinical isolates of A. baumannii, but not the common lab strain ATCC 19606 (19606), can persist and replicate in macrophages within spacious vacuoles that we called Acinetobacter Containing Vacuoles (ACV). In this work, we demonstrate that the modern A. baumannii clinical isolate 398, but not the lab strain 19606, can infect alveolar macrophages and produce ACVs in vivo in a murine pneumonia model. Both strains initially interact with the macrophage endocytic pathway, as indicated by EEA1 and LAMP1 markers; however, the fate of these strains diverges at a later stage. While 19606 is eliminated in an autophagy pathway, 398 replicates in ACVs and are not degraded. We show that 398 reverts the natural acidification of the phagosome by secreting large amounts of ammonia, a by-product of amino acid catabolism. We propose that this ability to survive within macrophages may be critical for the persistence of clinical A. baumannii isolates in the lung during a respiratory infection.

Polysialylation controls immune function of myeloid cells in murine model of pneumococcal pneumonia

Polysialic acid (polySia) is a post-translational modification of a select group of cell-surface proteins that guides cellular interactions. As the overall impact of changes in expression of this glycan on leukocytes during infection is not known, we evaluate the immune response of polySia-deficient ST8SiaIV-/- mice infected with Streptococcus pneumoniae (Spn). Compared with wild-type (WT) mice, ST8SiaIV-/- mice are less susceptible to infection and clear Spn from airways faster, with alveolar macrophages demonstrating greater viability and phagocytic activity. Leukocyte pulmonary recruitment, paradoxically, is diminished in infected ST8SiaIV-/- mice, corroborated by adoptive cell transfer, microfluidic migration experiments, and intravital microscopy, and possibly explained by dysregulated ERK1/2 signaling. PolySia is progressively lost from neutrophils and monocytes migrating from bone marrow to alveoli in Spn-infected WT mice, consistent with changing cellular functions. These data highlight multidimensional effects of polySia on leukocytes during an immune response and suggest therapeutic interventions for optimizing immunity.

Copper ions inhibit pentose phosphate pathway function in Staphylococcus aureus.

To gain a better insight of how Copper (Cu) ions toxify cells, metabolomic analyses were performed in S. aureus strains that lacks the described Cu ion detoxification systems (ΔcopBL ΔcopAZ; cop-). Exposure of the cop- strain to Cu(II) resulted in an increase in the concentrations of metabolites utilized to synthesize phosphoribosyl diphosphate (PRPP). PRPP is created using the enzyme phosphoribosylpyrophosphate synthetase (Prs) which catalyzes the interconversion of ATP and ribose 5-phosphate to PRPP and AMP. Supplementing growth medium with metabolites requiring PRPP for synthesis improved growth in the presence of Cu(II). A suppressor screen revealed that a strain with a lesion in the gene coding adenine phosphoribosyltransferase (apt) was more resistant to Cu. Apt catalyzes the conversion of adenine with PRPP to AMP. The apt mutant had an increased pool of adenine suggesting that the PRPP pool was being redirected. Over-production of apt, or alternate enzymes that utilize PRPP, increased sensitivity to Cu(II). Increasing or decreasing expression of prs resulted in decreased and increased sensitivity to growth in the presence of Cu(II), respectively. We demonstrate that Prs is inhibited by Cu ions in vivo and in vitro and that treatment of cells with Cu(II) results in decreased PRPP levels. Lastly, we establish that S. aureus that lacks the ability to remove Cu ions from the cytosol is defective in colonizing the airway in a murine model of acute pneumonia, as well as the skin. The data presented are consistent with a model wherein Cu ions inhibits pentose phosphate pathway function and are used by the immune system to prevent S. aureus infections.

Pseudomonas aeruginosapulmonary infection results in S100A8/9-dependent cardiac dysfunction

Abstract Pneumonia in ICU patients carries a bleak prognosis with high risk for cardiac dysfunction, largely due to cardiac infection and inflammation. Pseudomonas aeruginosa (P.a.) infection accounts nearly 20% of all cases of hospital acquired pneumonia with mortality rates >30%. P.a. infection induces a robust inflammatory response, which ideally enhances bacterial clearance. Unfortunately, excessive inflammation can also have negative effects, and often leads to cardiac dysfunction with associated morbidity and mortality. However, it remains unclear how P.a. lung infection causes cardiac dysfunction. Using a murine pneumonia model, we found that of P.a. infection of lungs led to severe cardiac left ventricular dysfunction and electrical abnormalities. More specifically, we found that neutrophil recruitment and release of S100A8/A9 in the lungs activates the TLR4/RAGE signaling pathways, which in turn enhance systemic inflammation and subsequent cardiac dysfunction. Paradoxically, global deletion of S100A8/A9 did not improve but aggravated cardiac dysfunction and immortality likely due to uncontrolled bacterial burden in the lungs and heart. Our results indicate that P.a. infection induced release of S100A8/9 is double-edged, providing increased risk for cardiac dysfunction yet limiting P.a. growth. Funding: NIH grants R01-AI 146252 and R21-AG 127783 Funding: NIH grants R01-AI 146252 and R21-AG 127783

Emergence of hypervirulent Pseudomonas aeruginosa pathotypically armed with co-expressed T3SS effectors ExoS and ExoU

Pseudomonas aeruginosa is a significant pathogen mainly causing healthcare-associated infections (HAIs). Newly emerging high-risk clones of P. aeruginosa with elevated virulence profiles furtherly cause severe community-acquired infections (CAIs). Usually, it is not common for P. aeruginosa to co-carry exoU and exoS genes, encoding two type III secretion system (T3SS) effectors. The pathogenicity mechanism of exoS+/exoU+ strains of P. aeruginosa remains unclear. Here, we provide detailed evidence for a subset of hypervirulent P. aeruginosa strains, which abundantly co-express and secrete the T3SS effectors ExoS and ExoU. The exoS+/exoU+P. aeruginosa strains were available to cause both HAIs and CAIs. The CAI-associated strains could elicit severe inflammation and hemorrhage, leading to higher death rates in a murine acute pneumonia model, and had great virulence potential in establishing chronic infections, demonstrating hypervirulence when compared to PAO1 (exoS+/exoU-) and PA14 (exoS-/exoU+). Both ExoS and ExoU were co-expressed and co-secreted in abundance in exoS+/exoU+ strains. Their abundant protein secretion could boost exoS+/exoU+ strains’ potentials for cytotoxicity in vitro and pathogenicity in vivo. Genomic evidence indicates that exoU acquisition is likely mediated by horizontal gene transfer (HGT) of the pathogenicity island PAPI-2, while deletion of exoU was sufficient to mitigate virulence in the exoS+/exoU+ strains. Furthermore, bioinformatics analysis showed that such exoS+/exoU+P. aeruginosa strains turned out to be widely distributed across the globe. Overall, the research provide detailed evidence for the high virulence and epidemicity of exoS+/exoU+ strains of P. aeruginosa, highlighting an urgent need for surveillance against these high-risk hypervirulent strains.

Naturally-occurring serotype 3 Streptococcus pneumoniae strains that lack functional pneumolysin and autolysin have attenuated virulence but induce localized protective immune responses.

Streptococcus pneumoniae is an important cause of fatal pneumonia in humans. These bacteria express virulence factors, such as the toxins pneumolysin and autolysin, that drive host inflammatory responses. In this study we confirm loss of pneumolysin and autolysin function in a group of clonal pneumococci that have a chromosomal deletion resulting in a pneumolysin-autolysin fusion gene Δ(lytA'-ply')593. The Δ(lytA'-ply')593 pneumococci strains naturally occur in horses and infection is associated with mild clinical signs. Here we use immortalized and primary macrophage in vitro models, which include pattern recognition receptor knock-out cells, and a murine acute pneumonia model to show that a Δ(lytA'-ply')593 strain induces cytokine production by cultured macrophages, however, unlike the serotype-matched ply+lytA+ strain, it induces less tumour necrosis factor α (TNFα) and no interleukin-1β production. The TNFα induced by the Δ(lytA'-ply')593 strain requires MyD88 but, in contrast to the ply+lytA+ strain, is not reduced in cells lacking TLR2, 4 or 9. In comparison to the ply+lytA+ strain in a mouse model of acute pneumonia, infection with the Δ(lytA'-ply')593 strain resulted in less severe lung pathology, comparable levels of interleukin-1α, but minimal release of other pro-inflammatory cytokines, including interferon-γ, interleukin-6 and TNFα. These results suggest a mechanism by which a naturally occurring Δ(lytA'-ply')593 mutant strain of S. pneumoniae that resides in a non-human host has reduced inflammatory and invasive capacity compared to a human S. pneumoniae strain. These data probably explain the relatively mild clinical disease in response to S. pneumoniae infection seen in horses in comparison to humans.

Staphylococcus aureus stimulates neutrophil itaconate production that suppresses the oxidative burst.

Neutrophils are critical in the host defense against Staphylococcus aureus, a major human pathogen. However, even in the setting of a robust neutrophil response, S.aureus can evade immune clearance. Here, we demonstrate that S.aureus impairs neutrophil function by triggering the production of the anti-inflammatory metabolite itaconate. The enzyme that synthesizes itaconate, Irg1, is selectively expressed in neutrophils during S.aureus pneumonia. Itaconate inhibits neutrophil glycolysis and oxidative burst, which impairs survival and bacterial killing. In a murine pneumonia model, neutrophil Irg1 expression protects the lung from excessive inflammation but compromises bacterial clearance. S.aureus is thus able to evade the innate immune response by targeting neutrophil metabolism and inducing the production of the anti-inflammatory metabolite itaconate.