Murine Model Of Acute Pneumonia Research Articles

Moderate Exercise Modulates Inflammatory Responses and Improves Survival in a Murine Model of Acute Pneumonia.

An association between physical inactivity and worse outcome during infectious disease has been reported. The effect of moderate exercise preconditioning on the immune response during an acute pneumonia in a murine model was evaluated. Laboratory experiments. C57BL6/j male mice. Six-week-old C57BL/6J mice were divided in two groups: an exercise group and a control group. In the exercise group, a moderate, progressive, and standardized physical exercise was applied for 8 weeks. It consisted in a daily treadmill training lasting 60 minutes and with an intensity of 65% of the maximal theoretical oxygen uptake. Usual housing recommendation were applied in the control group during the same period. After 8 weeks, pneumonia was induced in both groups by intratracheal instillation of a fixed concentration of a Klebsiella pneumoniae (5 × 103 colony-forming unit) solution. Mice preconditioned by physical exercise had a less sever onset of pneumonia as shown by a significant decrease of the Mouse Clinical Assessment Severity Score and had a significantly lower mortality compared with the control group (27% vs. 83%; p = 0.019). In the exercise group, we observed a significantly earlier but transient recruitment of inflammatory immune cells with a significant increase of neutrophils, CD4+ cells and interstitial macrophages counts compared with control group. Lung tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-10 were significantly decreased at 48 hours after pneumonia induction in the exercise group compared with the control group. In our model, preconditioning by moderate physical exercise improves outcome by reducing the severity of acute pneumonia with an increased but transient activation of the innate immune response.

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.

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.

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.

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.

Molecular Characterization of WCK 5222 (Cefepime/Zidebactam)-Resistant Mutants Developed from a Carbapenem-Resistant Pseudomonas aeruginosa Clinical Isolate.

ABSTRACTWCK 5222 (cefepime/zidebactam) is a β-lactam/β-lactamase inhibitor combination that is effective against a broad range of highly drug-resistant bacterial pathogens, including those producing metallo-β-lactamase. In this study, we isolated a multidrug-resistant Pseudomonas aeruginosa clinical strain that is resistant to a variety of β-lactam antibiotics and the ceftazidime-avibactam combination. A metallo-β-lactamase gene blaDIM-2 was identified on a self-transmissible megaplasmid in the strain, which confers the resistance to β-lactam antibiotics, leaving WCK 5222 potentially one of the last treatment resorts. In vitro passaging assay combined with whole-genome sequencing revealed mutations in the pbpA gene (encoding the zidebactam target protein PBP2) in the evolved resistant mutants. Among the mutations, a V516M mutation increased the bacterial virulence in a murine acute pneumonia model. Reconstitution of the mutations in the reference strain PAO1 verified their roles in the resistance to zidebactam and revealed their influences on cell morphology in the absence and presence of zidebactam. Microscale thermophoresis (MST) assays demonstrated that the mutations reduced the affinity between PBP2 and zidebactam to various extents. Overall, our results revealed that mutations in the pbpA gene might be a major cause of evolved resistance to WCK 5222 in clinical settings.IMPORTANCE Antibiotic resistance imposes a severe threat on human health. WCK 5222 is a β-lactam/β-lactamase inhibitor combination that is composed of cefepime and zidebactam. It is one of the few antibiotics in clinical trials that are effective against multidrug-resistant Pseudomonas aeruginosa, including those producing metallo-β-lactamases. Understanding the mechanisms and development of bacterial resistance to WCK 5222 may provide clues for the development of strategies to suppress resistant evolvement. In this study, we performed an in vitro passaging assay by using a multidrug-resistant P. aeruginosa clinical isolate. Our results revealed that mutations in the zidebactam target protein PBP2 play a major role in the bacterial resistance to WCK 5222. We further demonstrated that the mutations reduced the affinities between PBP2 and zidebactam and resulted in functional resistance of PBP2 to zidebactam.

SLy2-deficiency promotes B-1 cell immunity and triggers enhanced production of IgM and IgG2 antibodies against pneumococcal vaccine.

BackgroundDespite the benefits of existing vaccines, Streptococcus pneumoniae is still responsible for the greatest proportion of respiratory tract infections around the globe, thereby substantially contributing to morbidity and mortality in humans. B‐1 cells are key players of bacterial clearance during pneumococcal infection and even provide long‐lasting immunity towards S. pneumoniae. Previous reports strongly suggest an essential role of the immunoinhibitory adapter Src homology domain 3 lymphocyte protein 2 (SLy2) for B‐1 cell‐mediated antibody production. The objective of this study is to evaluate S. pneumoniae‐directed B cell responses in the context of SLy2 deficiency.MethodsB‐1 cell populations were analyzed via flow cytometry before and after pneumococcal immunization of SLy2‐deficient and wild‐type control mice. Global and vaccine‐specific immunoglobulin M (IgM) and IgG antibody titers were assessed by enzyme‐linked immunosorbent assay. To investigate survival rates during acute pneumococcal lung infection, mice were intranasally challenged with S. pneumoniae (serotype 3). Complementary isolated splenic B cells were stimulated in vitro and their proliferative response was assessed by fluorescent staining. In vitro antibody secretion was quantified by LEGENDplex.ResultsWe demonstrate increased frequencies of B‐1 cells and elevated titers of preantigenic IgM in SLy2‐deficient mice. In addition, these mice produce significantly more amounts of IgM and IgG2 upon pneumococcal vaccination. Knocking out SLy2 did not induce survival advantages in our murine model of acute pneumonia, indicating the presence of compensatory mechanisms.ConclusionOur results reveal reinforced specific antibody responses towards pneumococcal polysaccharides and enhanced IgG2 secretion as a consequence of SLy2 deficiency, which could be relevant to the development of more efficient vaccines.

The ClpXP Protease Contributes to Staphylococcus aureus Pneumonia

Staphylococcus aureus is a leading cause of pneumonia. We show here that the ClpXP protease involved in protein turnover is important for pathogenesis in a murine model of acute pneumonia. Staphylococcus aureus lacking this protease is attenuated in vivo, being rapidly cleared from the airway and leading to decreased immune cell influx and inflammation. Characterization of defined mutations in vitro identified defects in intracellular survival and protection against neutrophil killing. Our results further expand on what is known about ClpXP in the pathogenesis of S. aureus to include the respiratory tract.

Th17 responses to Pseudomonas aeruginosa vaccines are defective in cystic fibrosis mice

Abstract Pseudomonas aeruginosa causes chronic lung infections in people with cystic fibrosis (CF) associated with lung function decline. Since levels of IL-17 are high in the CF airway, it is unclear whether vaccines that induce Th17 responses in CF hosts would be effective, or potentially harmful. Our prior work showed that vaccine-induced Th17 cells confer antibody-independent protection against acute P. aeruginosa pneumonia in mice and that the P. aeruginosa protein PopB stimulates protective Th17 responses. In this study, we tested whether vaccine-induced Th17 responses are abnormal in CF mice. CF mice (delF508 in FVB/N background) and littermate controls were immunized intranasally either with live-attenuated P. aeruginosa vaccines or with PopB mixed with curdlan as adjuvant. We tested Th17 responses by measuring IL-17 secretion from splenocytes stimulated with antigens in vitro. Protective efficacy was assessed using a murine acute pneumonia model. Our results demonstrate that CF mice show 2-fold lower IL-17 recall responses in the spleen after IN immunization with a live-attenuated vaccine in comparison to wild-type mice (P<0.05 by ANOVA). CF mice were not protected when challenged with a LPS-heterologous strain, suggesting the absence of Th17-mediated immunity when LPS-directed opsonic antibodies are not available. CF mice immunized with PopB also showed defective Th17 responses in the spleen compared to wild-type or heterozygous mice (2-fold lower, P<0.05 by ANOVA). CF mice have low Th17 responses to P. aeruginosa vaccines. Correction of the underlying CF defect may be needed to achieve vaccine-induced protection against P. aeruginosa in the setting of CF.

Vancomycin intermediate strains of Staphylococcus aureus have dampened induction of type I interferon production

Abstract Studies by our group have shown that interferon (IFN) signaling contributes to pathogenesis of Staphylococcus aureus pneumonia and that different strains of S. aureus can activate type I IFN signaling via distinct receptors, such as TLR9 and NOD2, at various amplitudes. In this study we investigated the capacity of a wide range of S. aureus strains to induce both type I & III IFN. We screened 78 S. aureus strains from various clinical scenarios for their ability to induce IFN-β and IFN-λ in bone marrow-derived dendritic cells (BMDC) and found that both cytokines are differentially induced by various S. aureus strains independently of their isolation sites or methicillin resistance profiles. These induction patterns persisted over time and type I & III IFN differentially correlated with tumor necrosis factor α (TNF-α) production. Furthermore, vancomycin intermediate S. aureus (VISA) strains induced up to 45-fold less (P<0.001) IFN-β than the current epidemic methicillin-resistant S. aureus strain USA300 and displayed increased in vitro survival (up to 9-fold) after 4-hour exposure to BMDC. Further investigation of one VISA strain, HIP07930, showed decreased autolysis and increased resistance to lysostaphin compared to USA300, consistent with its reduction in IFN β induction. Interestingly, this strain also displayed decreased in vivo virulence compared to USA300 in a murine acute pneumonia model of infection (4.9-fold and 54.5-fold decrease in colony forming units in bronchoalveolar lavage fluid and lung homogenates, respectively, 24 hours post infection). Overall, this study demonstrates the heterogeneity of IFN induction by S. aureus and uncovered a unique property of VISA strains in their inability to induce type I IFN production.

Biological sex influences susceptibility to Acinetobacter baumannii pneumonia in mice.

Acinetobacter baumannii (A. baumannii) is an extremely versatile multidrug-resistant pathogen with a very high mortality rate; therefore, it has become crucial to understand the host response during its infection. Given the importance of mice for modeling infection and their role in preclinical drug development, equal emphasis should be placed on the use of both sexes. Through our studies using a murine model of acute pneumonia with A. baumannii, we observed that female mice were more susceptible to infection. Likewise, treatment of male mice with estradiol increased their susceptibility to infection. Analysis of the airway compartment revealed enhanced inflammation and reduced neutrophil and alveolar macrophage numbers compared with male mice. Depletion of either neutrophils or alveolar macrophages was important for bacterial clearance; however, depletion of alveolar macrophages further exacerbated female susceptibility because of severe alterations in metabolic homeostasis. Our data highlight the importance of using both sexes when assessing host immune pathways.

Interleukin-22 regulates interferon lambda expression in a mice model of pseudomonas aeruginosa pneumonia

BackgroundInterleukin (IL)-22 is a cytokine involved in tissue protection and repair following lung pathologies. Interferon (IFN)-λ cytokines displayed similar properties during viral infection and a synergy of action between these two players has been documented in the intestine. We hypothesize that during Pseudomonas aeruginosa challenge, IL-22 up-regulates IFN-λ and that IFN-λ exhibits protective functions during Pseudomonas aeruginosa acute pneumonia model in mice. MethodsUsing an in vitro human alveolar epithelial cell line A549, we assessed the ability of IL-22 to enhance IFN-λ expression during infection. IFN-λ protective function was evaluated in an acute mouse pneumonia model. ResultsWe first demonstrated in murine lungs that only type-II alveolar cells express IL-22 receptor and that IL-22 treatment of A549 cell line up-regulates IFN-λ expression. In a murine acute pneumonia model, IL-22 administration maintained significant IFN-λ levels in the broncho-alveolar fluids whereas IL-22 neutralization abolished IFN-λ up-regulation. In vivo administration of IFN-λ during Pseudomonas aeruginosa pneumonia improves mice outcome by dampening neutrophil recruitment and decreasing epithelium damages. DiscussionWe show here that IL-22 regulates IFN-λ levels during Pseudomonas aeruginosa pneumonia.

Tanshinones: First-in-Class Inhibitors of the Biogenesis of the Type 3 Secretion System Needle of Pseudomonas aeruginosa for Antibiotic Therapy.

The type 3 secretion system (T3SS) found as cell-surface appendages of many pathogenic Gram-negative bacteria, although nonessential for bacterial survival, is an important therapeutic target for drug discovery and development aimed at inhibiting bacterial virulence without inducing antibiotic resistance. We designed a fluorescence-polarization-based assay for high-throughput screening as a mechanistically well-defined general strategy for antibiotic discovery targeting the T3SS and made a serendipitous discovery of a subset of tanshinones—natural herbal compounds in traditional Chinese medicine widely used for the treatment of cardiovascular and cerebrovascular diseases—as effective inhibitors of the biogenesis of the T3SS needle of multi-drug-resistant Pseudomonas aeruginosa. By inhibiting the T3SS needle assembly and, thus, cytotoxicity and pathogenicity, selected tanshinones reduced the secretion of bacterial virulence factors toxic to macrophages in vitro, and rescued experimental animals challenged with lethal doses of Pseudomonas aeruginosa in a murine model of acute pneumonia. As first-in-class inhibitors with a demonstrable safety profile in humans, tanshinones may be used directly to alleviate Pseudomonas-aeruginosa-associated pulmonary infections without inducing antibiotic resistance. Since the T3SS is highly conserved among Gram-negative bacteria, this antivirulence strategy may be applicable to the discovery and development of novel classes of antibiotics refractory to existing resistance mechanisms for the treatment of many bacterial infections.

Construction of a Protective Vaccine Against Lipopolysaccharide-Heterologous Pseudomonas aeruginosa Strains Based on Expression Profiling of Outer Membrane Proteins During Infection.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen, which causes infectious disease in patients with cystic fibrosis and compromised immunity. P. aeruginosa is difficult to eradicate because of its intrinsic resistance to most traditional antibiotics as well as acquired resistance mechanisms after decades of antibiotic usage. A full understanding of the P. aeruginosa pathogenesis mechanisms is necessary for the development of novel prevention and treatment strategies. To identify novel vaccine candidates, here we comprehensively examined the expression levels of all the known outer membrane proteins in two P. aeruginosa strains in a murine acute pneumonia model. OprH was one of the most highly expressed proteins during infection. In addition, OprH is known to be highly immunogenic and accessible by host proteins. Thus, it was chosen as a vaccine candidate. To further identify vaccine candidates, 34 genes highly expressed during infection were evaluated for their contributions in virulence by testing individual transposon insertion mutants. Among them, fpvA, hasR, and foxA were found essential for bacterial virulence and therefore included in vaccine construction. Immunization with a mixture of FpvA, HasR, and FoxA rendered no protection, however, while immunization by OprH refolded in liposomes elicited specific opsonic antibodies and conferred protection against two lipopolysaccharide-heterologous P. aeruginosa strains (PA14 and PA103). Overall, by studying the expression profile of the P. aeruginosa outer membrane proteins during infection, we identified OprH as a potential vaccine candidate for the prevention of lung infection by P. aeruginosa.

Pulmonary Surfactant Promotes Virulence Gene Expression and Biofilm Formation in Klebsiella pneumoniae.

The interactions between Klebsiella pneumoniae and the host environment at the site of infection are largely unknown. Pulmonary surfactant serves as an initial point of contact for inhaled bacteria entering the lung and is thought to contain molecular cues that aid colonization and pathogenesis. To gain insight into this ecological transition, we characterized the transcriptional response of K. pneumoniae MGH 78578 to purified pulmonary surfactant. This work revealed changes within the K. pneumoniae transcriptome that likely contribute to host colonization, adaptation, and virulence in vivo Notable transcripts expressed under these conditions include genes involved in capsule synthesis, lipopolysaccharide modification, antibiotic resistance, biofilm formation, and metabolism. In addition, we tested the contributions of other surfactant-induced transcripts to K. pneumoniae survival using engineered isogenic KPPR1 deletion strains in a murine model of acute pneumonia. In these infection studies, we identified the MdtJI polyamine efflux pump and the ProU glycine betaine ABC transporter to be significant mediators of K. pneumoniae survival within the lung and confirmed previous evidence for the importance of de novo leucine synthesis to bacterial survival during infection. Finally, we determined that pulmonary surfactant promoted type 3 fimbria-mediated biofilm formation in K. pneumoniae and identified two surfactant constituents, phosphatidylcholine and cholesterol, that drive this response. This study provides novel insight into the interactions occurring between K. pneumoniae and the host at an important infection site and demonstrates the utility of purified lung surfactant preparations for dissecting host-lung pathogen interactions in vitro.

PutA Is Required for Virulence and Regulated by PruR in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogenic bacterium, causes acute and chronic infections. Upon entering the host, P. aeruginosa alters global gene expression to adapt to host environment and avoid clearance by the host immune system. Proline utilization A (PutA) is a bifunctional enzyme, which converts proline to glutamate. Here we report that PutA was required for the virulence of P. aeruginosa in a murine acute pneumonia model. A putA mutant was more susceptible to oxidative stress compared to the wild type strain. An AraC/XylS family protein, PruR, directly bound to the upstream of −35 box in the putA promoter and activated putA expression. High concentration of proline in bacteria up-regulated pruR expression, which led to the activation of putA expression. As a feedback regulation, glutamate produced by PutA released PruR from the putA promoter and turned off the putA expression. PruR affected bacterial virulence through the regulation of the putA expression. Altogether, these data are the first to reveal that PutA plays an important role in the pathogenesis of P. aeruginosa, as well as to describe the genetic regulation of PutA in P. aeruginosa.

Fis Regulates Type III Secretion System by Influencing the Transcription of exsA in Pseudomonas aeruginosa Strain PA14.

Fis is a versatile DNA binding protein in bacteria. It has been demonstrated in multiple bacteria that Fis plays crucial roles in regulating bacterial virulence factors and optimizing bacterial adaptation to various environments. However, the role of Fis in Pseudomonas aeruginosa virulence as well as gene regulation remains largely unknown. Here, we found that Fis was required for the virulence of P. aeruginosa in a murine acute pneumonia model. Transcriptome analysis revealed that expression of T3SS genes, including master regulator ExsA, was defective in a fis::Tn mutant. We further demonstrate that the continuous transcription of exsC, exsE, exsB, and exsA driven by the exsC promoter was required for the activation of T3SS. Fis was found to specifically bind to the exsB-exsA intergenic region and plays an essential role in the transcription elongation from exsB to exsA. Therefore, we found a novel role of Fis in the regulation of exsA expression.

Pseudomonas aeruginosa Enolase Influences Bacterial Tolerance to Oxidative Stresses and Virulence.

Pseudomonas aeruginosa is a Gram negative opportunistic pathogenic bacterium, which causes acute and chronic infections. Upon entering the host, bacteria alter global gene expression to adapt to host environment and avoid clearance by the host. Enolase is a glycolytic enzyme involved in carbon metabolism. It is also a component of RNA degradosome, which is involved in RNA processing and gene regulation. Here, we report that enolase is required for the virulence of P. aeruginosa in a murine acute pneumonia model. Mutation of enolase coding gene (eno) increased bacterial susceptibility to neutrophil mediated killing, which is due to reduced tolerance to oxidative stress. Catalases and alkyl hydroperoxide reductases play a major role in protecting the cell from oxidative damages. In the eno mutant, the expression levels of catalases (KatA and KatB) were similar as those in the wild type strain in the presence of H2O2, however, the expression levels of alkyl hydroperoxide reductases (AhpB and AhpC) were significantly reduced. Overexpression of ahpB but not ahpC in the eno mutant fully restored the bacterial resistance to H2O2 as well as neutrophil mediated killing, and partially restored bacterial virulence in the murine acute pneumonia model. Therefore, we have identified a novel role of enolase in the virulence of P. aeruginosa.