Detection of influenza virus and Streptococcus pneumoniae in air sampled from co-infected ferrets and analysis of their influence on pathogen stability.

Secondary infection with Streptococcus pneumoniae has contributed significantly to morbidity and mortality during multiple influenza virus pandemics and remains a common threat today. During a concurrent infection, both pathogens can influence the transmission of each other, but the mechanisms behind this are unclear. In this study, condensation air sampling and cyclone bioaerosol sampling were performed using ferrets first infected with the 2009 H1N1 pandemic influenza virus (H1N1pdm09) and secondarily infected with S. pneumoniae strain D39 (Spn). We detected viable pathogens and microbial nucleic acid in expelled aerosols from co-infected ferrets, suggesting that these microbes could be present in the same respiratory expulsions. To assess whether microbial communities impact pathogen stability within an expelled droplet, we performed experiments measuring viral and bacterial persistence in 1 µL droplets. We observed that H1N1pdm09 stability was unchanged in the presence of Spn. Further, Spn stability was moderately increased in the presence of H1N1pdm09, although the degree of stabilization differed between airway surface liquid collected from individual patient cultures. These findings are the first to collect both pathogens from the air and in doing so, they provide insight into the interplay between these pathogens and their hosts.IMPORTANCEThe impact of microbial communities on transmission fitness and environmental persistence is under-studied. Environmental stability of microbes is crucial to identifying transmission risks and mitigation strategies, such as removal of contaminated aerosols and decontamination of surfaces. Co-infection with S. pneumoniae is very common during influenza virus infection, but little work has been done to understand whether S. pneumoniae alters stability of influenza virus, or vice versa, in a relevant system. Here, we demonstrate that influenza virus and S. pneumoniae are expelled by co-infected hosts. Our stability assays did not reveal any impact of S. pneumoniae on influenza virus stability, but did show a trend towards increased stability of S. pneumoniae in the presence of influenza viruses. Future work characterizing environmental persistence of viruses and bacteria should include microbially complex solutions to better mimic physiologically relevant conditions.

Impact of Pneumococcal and Viral Pneumonia on the Respiratory and Intestinal Tract Microbiomes of Mice.

With 2.56 million deaths worldwide annually, pneumonia is one of the leading causes of death. The most frequent causative pathogens are Streptococcus pneumoniae and influenza A virus. Lately, the interaction between the pathogens, the host, and its microbiome have gained more attention. The microbiome is known to promote the immune response toward pathogens; however, our knowledge on how infections affect the microbiome is still scarce. Here, the impact of colonization and infection with S. pneumoniae and influenza A virus on the structure and function of the respiratory and gastrointestinal microbiomes of mice was investigated. Using a meta-omics approach, we identified specific differences between the bacterial and viral infection. Pneumococcal colonization had minor effects on the taxonomic composition of the respiratory microbiome, while acute infections caused decreased microbial complexity. In contrast, richness was unaffected following H1N1 infection. Within the gastrointestinal microbiome, we found exclusive changes in structure and function, depending on the pathogen. While pneumococcal colonization had no effects on taxonomic composition of the gastrointestinal microbiome, increased abundance of Akkermansiaceae and Spirochaetaceae as well as decreased amounts of Clostridiaceae were exclusively found during invasive S. pneumoniae infection. The presence of Staphylococcaceae was specific for viral pneumonia. Investigation of the intestinal microbiomés functional composition revealed reduced expression of flagellin and rubrerythrin and increased levels of ATPase during pneumococcal infection, while increased amounts of acetyl coenzyme A (acetyl-CoA) acetyltransferase and enoyl-CoA transferase were unique after H1N1 infection. In conclusion, identification of specific taxonomic and functional profiles of the respiratory and gastrointestinal microbiome allowed the discrimination between bacterial and viral pneumonia. IMPORTANCE Pneumonia is one of the leading causes of death worldwide. Here, we compared the impact of bacterial- and viral-induced pneumonia on the respiratory and gastrointestinal microbiome. Using a meta-omics approach, we identified specific profiles that allow discrimination between bacterial and viral causative.

CSF-contacting neurons respond to Streptococcus pneumoniae and promote host survival during central nervous system infection.

The pathogenic bacterium Streptococcus pneumoniae (S. pneumoniae) can invade the cerebrospinal fluid (CSF) and cause meningitis with devastating consequences. Whether and how sensory cells in the central nervous system (CNS) become activated during bacterial infection, as recently reported for the peripheral nervous system, is not known. We find that CSF infection by S. pneumoniae in larval zebrafish leads to changes in posture and behavior that are reminiscent of pneumococcal meningitis, including dorsal arching and epileptic-like seizures. We show that during infection, invasion of the CSF by S. pneumoniae massively activates in vivo sensory neurons contacting the CSF, referred to as "CSF-cNs" and previously shown to detect spinal curvature and to control posture, locomotion, and spine morphogenesis. We find that CSF-cNs express orphan bitter taste receptors and respond in vitro to bacterial supernatant and metabolites via massive calcium transients, similar to the ones observed in vivo during infection. Upon infection, CSF-cNs also upregulate the expression of numerous cytokines and complement components involved in innate immunity. Accordingly, we demonstrate, using cell-specific ablation and blockade of neurotransmission, that CSF-cN neurosecretion enhances survival of the host during S. pneumoniae infection. Finally, we show that CSF-cNs respond to various pathogenic bacteria causing meningitis in humans, as well as to the supernatant of cells infected by a neurotropic virus. Altogether, our work uncovers that central sensory neurons in the spinal cord, previously involved in postural control and morphogenesis, contribute as well to host survival by responding to the invasion of the CSF by pathogenic bacteria during meningitis.

Streptococcus pneumoniae Rapidly Translocate from the Nasopharynx through the Cribriform Plate to Invade the Outer Meninges.

The entry routes and translocation mechanisms of microorganisms or particulate materials into the central nervous system remain obscure We report here that Streptococcus pneumoniae (pneumococcus), or polystyrene microspheres of similar size, appear in the meninges of the dorsal cortex of mice within minutes of inhaled delivery. Recovery of viable bacteria from dissected tissue and fluorescence microscopy show that up to at least 72 h, pneumococci and microspheres were predominantly found in the outer of the two meninges: the pachymeninx. No pneumococci were found in blood or cerebrospinal fluid. Intravital imaging through the skull, aligned with flow cytometry showed recruitment and activation of LysM+ cells in the dorsal pachymeninx at 5 and 10 hours following intranasal infection. Imaging of the cribriform plate suggested that both pneumococci and microspheres entered through the foramina via an inward flow of fluid connecting the nose to the pachymeninx. Our findings bring new insight into the varied mechanisms of pneumococcal invasion of the central nervous system, but they are also pertinent to the delivery of drugs to the brain and the entry of airborne particulate matter into the cranium. IMPORTANCE Using two-photon imaging, we show that pneumococci translocate from the nasopharynx to the dorsal meninges of a mouse in the absence of any bacteria found in blood or cerebrospinal fluid. Strikingly, this takes place within minutes of inhaled delivery of pneumococci, suggesting the existence of an inward flow of fluid connecting the nasopharynx to the meninges, rather than a receptor-mediated mechanism. We also show that this process is size dependent, as microspheres of the same size as pneumococci can translocate along the same pathway, while larger size microspheres cannot. Furthermore, we describe the host response to invasion of the outer meninges. Our study provides a completely new insight into the key initial events that occur during the translocation of pneumococci directly from the nasal cavity to the meninges, with relevance to the development of intranasal drug delivery systems and the investigations of brain damage caused by inhaled air pollutants.

SARS-CoV-2 Spike Protein and Mouse Coronavirus Inhibit Biofilm Formation by Streptococcus pneumoniae and Staphylococcus aureus.

The presence of co-infections or superinfections with bacterial pathogens in COVID-19 patients is associated with poor outcomes, including increased morbidity and mortality. We hypothesized that SARS-CoV-2 and its components interact with the biofilms generated by commensal bacteria, which may contribute to co-infections. This study employed crystal violet staining and particle-tracking microrheology to characterize the formation of biofilms by Streptococcus pneumoniae and Staphylococcus aureus that commonly cause secondary bacterial pneumonia. Microrheology analyses suggested that these biofilms were inhomogeneous soft solids, consistent with their dynamic characteristics. Biofilm formation by both bacteria was significantly inhibited by co-incubation with recombinant SARS-CoV-2 spike S1 subunit and both S1 + S2 subunits, but not with S2 extracellular domain nor nucleocapsid protein. Addition of spike S1 and S2 antibodies to spike protein could partially restore bacterial biofilm production. Furthermore, biofilm formation in vitro was also compromised by live murine hepatitis virus, a related beta-coronavirus. Supporting data from LC-MS-based proteomics of spike-biofilm interactions revealed differential expression of proteins involved in quorum sensing and biofilm maturation, such as the AI-2E family transporter and LuxS, a key enzyme for AI-2 biosynthesis. Our findings suggest that these opportunistic pathogens may egress from biofilms to resume a more virulent planktonic lifestyle during coronavirus infections. The dispersion of pathogens from biofilms may culminate in potentially severe secondary infections with poor prognosis. Further detailed investigations are warranted to establish bacterial biofilms as risk factors for secondary pneumonia in COVID-19 patients.

Loop 422-437 in NanA from Streptococcus pneumoniae plays the role of an active site lid and is associated with allosteric regulation.

Neuraminidase A from Streptococcus pneumoniae (NanA) is considered a potentially key pathogenicity factor and a promising drug target to treat human infectious diseases. Computational and experimental efforts are increasingly being used to study its structure and function which yet remain poorly understood. In this work, we characterized structural dynamics of NanA's active site and gained novel mechanistic insights into its implications for a ligand binding. We based our study on supercomputer modeling and bioinformatic analysis with a help of crystallographic data and by bringing together previously published experimental data. The most prominent conformational plasticity was observed in the loop 422-437, accompanied by the mobility of adjacent loops 352-360 and 579-587. These structural elements had been undergoing spontaneous fluctuations apparently playing the role of an active site lid: an "open" state allowed substrate access to the active site, while a "closed" state accommodated the substrate in a catalytically favorable orientation. We observed that conformational plasticity of the loop 422-437 promoted the formation of an additional pocket located between catalytic and insertion domains of the enzyme. We recently argued this site was able to bind isoprenylated flavone artocarpin as an inhibitor of pneumococcal biofilm formation. Here we showed that accommodation of the mixed-type inhibitor artocarpin in this pocket limited mobility of the loop 422-437. This represents a plausible explanation of artocarpin's regulatory effect on the enzyme's catalytic function which seems to be independent of its role in preventing biofilm formation.

IL-4 plays an essential role in DnaJ-ΔA146Ply-mediated immunoprotection against Streptococcus pneumoniae in mice.

The fusion protein DnaJ-ΔA146Ply is protective against pneumococcal infections in mice. However, we found that immunized IL-4-/- mice showed significant lower survival rates and higher bacterial loads than did wild-type (WT) mice after being challenged. We explored the role of IL-4 in the protective immunity conferred by DnaJ-ΔA146Ply. Our results showed that there were no significant differences in antibody titers between immunized WT mice and IL-4-/- mice. The bacterial loads of passively immunized IL-4-/- mice were significantly higher than those of WT mice, while mice immunized with anti-DnaJ-ΔA146Ply serum from WT and IL-4-/- mice showed similar capacity for bacterial clearance. DnaJ-ΔA146Ply-dependent phagocytosis of IL-4-/- neutrophils was significant decreased compared with that of WT neutrophils. The levels of Syk and phosphor-Syk in IL-4-/- neutrophils were decreased compared with those in WT neutrophils. Additionally, Splenocytes in IL-4-/- mice triggered significantly higher levels of IFN-γ and IL-17A than did splenocytes in WT mice. Taken together, our findings illustrate that IL-4 deficiency does not influence the antibody production or antibody effect, but change the cellular immune response induced by DnaJ-ΔA146Ply. Additionally, IL-4 can enhance the antibody-dependent phagocytosis of neutrophils partially by activating Syk and participate in the protective immunity induced by DnaJ-ΔA146Ply.

Pandemic Influenza Infection Promotes Streptococcus pneumoniae Infiltration, Necrotic Damage, and Proteomic Remodeling in the Heart.

For over a century, it has been reported that primary influenza infection promotes the development of a lethal form of bacterial pulmonary disease. More recently, pneumonia events caused by both viruses and bacteria have been directly associated with cardiac damage. Importantly, it is not known whether viral-bacterial synergy extends to extrapulmonary organs such as the heart. Using label-free quantitative proteomics and molecular approaches, we report that primary infection with pandemic influenza A virus leads to increased Streptococcus pneumoniae translocation to the myocardium, leading to general biological alterations. We also observed that each infection alone led to proteomic changes in the heart, and these were exacerbated in the secondary bacterial infection (SBI) model. Gene ontology analysis of significantly upregulated proteins showed increased innate immune activity, oxidative processes, and changes to ion homeostasis during SBI. Immunoblots confirmed increased complement and antioxidant activity in addition to increased expression of angiotensin-converting enzyme 2. Using an in vitro model of sequential infection in human cardiomyocytes, we observed that influenza enhances S. pneumoniae cytotoxicity by promoting oxidative stress enhancing bacterial toxin-induced necrotic cell death. Influenza infection was found to increase receptors that promote bacterial adhesion, such as polymeric immunoglobulin receptor and fibronectin leucine-rich transmembrane protein 1 in cardiomyocytes. Finally, mice deficient in programmed necrosis (i.e., necroptosis) showed enhanced innate immune responses, decreased virus-associated pathways, and promotion of mitochondrial function upon SBI. The presented results provide the first in vivo evidence that influenza infection promotes S. pneumoniae infiltration, necrotic damage, and proteomic remodeling of the heart. IMPORTANCE Adverse cardiac events are a common complication of viral and bacterial pneumonia. For over a century, it has been recognized that influenza infection promotes severe forms of pulmonary disease mainly caused by the bacterium Streptococcus pneumoniae. The extrapulmonary effects of secondary bacterial infections to influenza virus are not known. In the present study, we used a combination of quantitative proteomics and molecular approaches to assess the underlying mechanisms of how influenza infection promotes bacteria-driven cardiac damage and proteome remodeling. We further observed that programmed necrosis (i.e., necroptosis) inhibition leads to reduced damage and proteome changes associated with health.

Differential Ubiquitination as an Effective Strategy Employed by the Blood-Brain Barrier for Prevention of Bacterial Transcytosis.

The protective mechanisms of blood-brain barrier (BBB) prohibiting entry of pathogens into central nervous system (CNS) are critical for maintenance of brain homeostasis. These include various intracellular defense mechanisms that are vital to block transcytosis of neurotropic pathogens into the CNS. However, mechanistic details of coordination between these defense pathways remain unexplored. In this study, we established that BBB-driven ubiquitination acts as a major intracellular defense mechanism for clearance of Streptococcus pneumoniae, a critical neurotropic pathogen, during transit through BBB. Our findings suggest that the BBB employs differential ubiquitination with either K48- or K63-ubiquitin (Ub) chain topologies as an effective strategy to target S. pneumoniae toward diverse killing pathways. While K63-Ub decoration triggers autophagic killing, K48-Ub directs S. pneumoniae exclusively toward proteasomes. Time-lapse fluorescence imaging involving proteasomal marker LMP2 revealed that in the BBB, the majority of the ubiquitinated S. pneumoniae was cleared by proteasome. Fittingly, inhibition of proteasome and autophagy pathway led to accumulation of K48-Ub- and K63-Ub-marked S. pneumoniae, respectively, and triggered significant increases in intracellular S. pneumoniae burden. Moreover, genetic impairment of either K48- or K63-Ub chain formation demonstrated that although both chain types are key in disposal of intracellular S. pneumoniae, K48-Ub chains and subsequent proteasomal degradation have more pronounced contributions to intracellular S. pneumoniae killing in the BBB. Collectively, these observations, for the first time, illustrated a pivotal role of differential ubiquitination deployed by BBB in orchestrating a symphony of intracellular defense mechanisms for interception and degradation of S. pneumoniae, blocking its entry into the brain, which could be exploited to prevent bacterial CNS infections. IMPORTANCE The blood-brain barrier (BBB) represents a unique cellular barrier that provides structural integrity and protection to the CNS from pathogen invasion. Recently, ubiquitination, which is key for cellular homeostasis, was shown to be involved in pathogen clearance. In this study, we deciphered that the BBB deploys differential ubiquitination as an effective strategy to prevent S. pneumoniae trafficking into the brain. The different ubiquitin chain topologies formed on S. pneumoniae dictated the selection of downstream degradative pathways, namely, autophagy and proteasomes, among which the contribution of the proteasomal system in S. pneumoniae killing is more pronounced. Overall our study revealed how the BBB deploys differential ubiquitination as a strategy for synchronization of various intracellular defense pathways, which work in tandem to ensure the brain's identity as an immunologically privileged site.

Anatomical Site-Specific Carbohydrate Availability Impacts Streptococcus pneumoniae Virulence and Fitness during Colonization and Disease.

Streptococcus pneumoniae colonizes the nasopharynx asymptomatically but can also cause severe life-threatening disease. Importantly, stark differences in carbohydrate availability exist between the nasopharynx and invasive disease sites, such as the bloodstream, which most likely impact S. pneumoniae's behavior. Herein, using chemically defined medium (CDM) supplemented with physiological levels of carbohydrates, we examined how anatomical site-specific carbohydrate availability impacted S. pneumoniae physiology and virulence. S. pneumoniae cells grown in CDM modeling the nasopharynx (CDM-N) had reduced metabolic activity and a lower growth rate, demonstrated mixed acid fermentation with marked H2O2 production, and were in a carbon-catabolite repression (CCR)-derepressed state versus S. pneumoniae cells grown in CDM modeling blood (CDM-B). Using transcriptome sequencing (RNA-seq), we determined the transcriptome for the S. pneumoniae wild-type (WT) strain and its isogenic CCR-deficient mutant in CDM-N and CDM-B. Genes with altered expression as a result of changes in carbohydrate availability or catabolite control protein deficiency, respectively, were primarily involved in carbohydrate metabolism, but also encoded established virulence determinants, such as polysaccharide capsule and surface adhesins. We confirmed that anatomical site-specific carbohydrate availability directly influenced established S. pneumoniae virulence traits. S. pneumoniae cells grown in CDM-B formed shorter chains, produced more capsule, were less adhesive, and were more resistant to macrophage killing in an opsonophagocytosis assay. Moreover, growth of S. pneumoniae in CDM-N or CDM-B prior to the challenge of mice impacted relative fitness in a colonization model and invasive disease model, respectively. Thus, anatomical site-specific carbohydrate availability alters S. pneumoniae physiology and virulence, in turn promoting anatomical site-specific fitness.

Comparison between the molecular diagnostic test and chest X-ray combined with multi-slice spiral CT in the diagnosis of lobar pneumonia.

Lobar pneumonia is an inflammatory condition of the lung that mainly affects the lobes of the lungs and the alveoli, and it is usually caused by a bacterial infection. There are many ways to diagnosis this disease. But an early and accurate method for lobar pneumonia diagnosis has an important role in its treatment. Therefore, in this study, a comparison between the molecular diagnostic test and chest x-ray combined with multi-slice spiral CT was done to find out better diagnosis of lobar pneumonia. For this purpose, 122 individuals suspected of lobar pneumonia were studied by clinical examination, chest X-ray, and multi-slice spiral CT. For the molecular diagnosis test, the multiplex PCR was used for two main causes of the disease, Streptococcus pneumoniae and Klebsiella pneumoniae. Results showed that the specificity for Chest X-ray + Multi-slice Spiral CT had the highest amount (82.8%), but high sensitivity (100%) belonged to a molecular diagnostic test for both bacteria. On the other hand, the sensitivity and specificity of Streptococcus pneumoniae were better than Klebsiella pneumoniae and the possibility of error in Streptococcus pneumoniae was lower than Klebsiella pneumoniae. In general, although the Chest X-ray + Multi-slice Spiral CT method was better than the molecular diagnosis test, it could not identify the causative agent and did not show a difference between pathogens for better antibiotic treatment, and also the possibility of diagnosis is low at the beginning of the disease. Therefore, according to the results of the current study, the best way to diagnose lobar pneumonia is to use both methods, simultaneously.

Porphyromonas gingivalis Components/Secretions Synergistically Enhance Pneumonia Caused by Streptococcus pneumoniae in Mice.

Streptococcus pneumoniae is an important causative organism of respiratory tract infections. Although periodontal bacteria have been shown to influence respiratory infections such as aspiration pneumonia, the synergistic effect of S. pneumoniae and Porphyromonas gingivalis, a periodontopathic bacterium, on pneumococcal infections is unclear. To investigate whether P. gingivalis accelerates pneumococcal infections, we tested the effects of inoculating P. gingivalis culture supernatant (PgSup) into S. pneumoniae-infected mice. Mice were intratracheally injected with S. pneumoniae and PgSup to induce pneumonia, and lung histopathological sections and the absolute number and frequency of neutrophils and macrophages in the lung were analyzed. Proinflammatory cytokine/chemokine expression was examined by qPCR and ELISA. Inflammatory cell infiltration was observed in S. pneumoniae-infected mice and S. pnemoniae and PgSup mixed-infected mice, and mixed-infected mice showed more pronounced inflammation in lung. The ratios of monocytes/macrophages and neutrophils were not significantly different between the lungs of S. pneumoniae-infected mice and those of mixed-infected mice. PgSup synergistically increased TNF-α expression/production and IL-17 production compared with S. pneumoniae infection alone. We demonstrated that PgSup enhanced inflammation in pneumonia caused by S. pneumoniae, suggesting that virulence factors produced by P. gingivalis are involved in the exacerbation of respiratory tract infections such as aspiration pneumonia.

Cell Wall Stress Stimulates the Activity of the Protein Kinase StkP of Streptococcus pneumoniae, Leading to Multiple Phosphorylation.

Streptococcus pneumoniae is an opportunistic human pathogen that encodes a single eukaryotic-type Ser/Thr protein kinase StkP and its functional counterpart, the protein phosphatase PhpP. These signaling enzymes play critical roles in coordinating cell division and growth in pneumococci. In this study, we determined the proteome and phosphoproteome profiles of relevant mutants. Comparison of those with the wild-type provided a representative dataset of novel phosphoacceptor sites and StkP-dependent substrates. StkP phosphorylates key proteins involved in cell division and cell wall biosynthesis in both the unencapsulated laboratory strain Rx1 and the encapsulated virulent strain D39. Furthermore, we show that StkP plays an important role in triggering an adaptive response induced by a cell wall-directed antibiotic. Phosphorylation of the sensor histidine kinase WalK and downregulation of proteins of the WalRK core regulon suggest crosstalk between StkP and the WalRK two-component system. Analysis of proteomic profiles led to the identification of gene clusters regulated by catabolite control mechanisms, indicating a tight coupling of carbon metabolism and cell wall homeostasis. The imbalance of steady-state protein phosphorylation in the mutants as well as after antibiotic treatment is accompanied by an accumulation of the global Spx regulator, indicating a Spx-mediated envelope stress response. In summary, StkP relays the perceived signal of cell wall status to key cell division and regulatory proteins, controlling the cell cycle and cell wall homeostasis.

Capsule Promotes Intracellular Survival and Vascular Endothelial Cell Translocation during Invasive Pneumococcal Disease.

The polysaccharide capsule that surrounds Streptococcus pneumoniae (Spn) is one of its most important virulence determinants, serving to protect against phagocytosis. To date, 100 biochemical and antigenically distinct capsule types, i.e., serotypes, of Spn have been identified. Yet how capsule influences pneumococcal translocation across vascular endothelial cells (VEC), a key step in the progression of invasive disease, was unknown. Here, we show that despite capsule being inhibitory of Spn uptake by VEC, capsule enhances the escape rate of internalized pneumococci and thereby promotes translocation. Upon investigation, we determined that capsule protected Spn against intracellular killing by VEC and H2O2-mediated killing in vitro. Using a nitroblue tetrazolium reduction assay and nuclear magnetic resonance (NMR) analyses, purified capsule was confirmed as having antioxidant properties which varied according to serotype. Using an 11-member panel of isogenic capsule-switch mutants, we determined that serotype affected levels of Spn resistance to H2O2-mediated killing in vitro, with killing resistance correlated positively with survival duration within VEC, rate of transcytosis to the basolateral surface, and human attack rates. Experiments with mice supported our in vitro findings, with Spn producing oxidative-stress-resistant type 4 capsule being more organ-invasive than that producing oxidative-stress-sensitive type 2 capsule during bacteremia. Capsule-mediated protection against intracellular killing was also observed for Streptococcus pyogenes and Staphylococcus aureus. We conclude that capsular polysaccharide plays an important role within VEC, serving as an intracellular antioxidant, and that serotype-dependent differences in antioxidant capabilities impact the efficiency of VEC translocation and a serotype's potential for invasive disease. IMPORTANCE Streptococcus pneumoniae (Spn) is the leading cause of invasive disease. Importantly, only a subset of the 100 capsule types carried by Spn cause the majority of serious infections, suggesting that the biochemical properties of capsular polysaccharide are directly tied to virulence. Here, we describe a new function for Spn's capsule-conferring resistance to oxidative stress. Moreover, we demonstrate that capsule promotes intracellular survival of pneumococci within vascular endothelial cells and thereby enhances bacterial translocation across the vasculature and into organs. Using isogenic capsule-switch mutants, we show that different capsule types, i.e., serotypes, vary in their resistance to oxidative stress-mediated killing and that resistance is positively correlated with intracellular survival in an in vitro model, organ invasion during bacteremia in vivo, and epidemiologically established pneumococcal attack rates in humans. Our findings define a new role of capsule and provide an explanation for why certain serotypes of Spn more frequently cause invasive pneumococcal disease.

Host Stress Signals Stimulate Pneumococcal Transition from Colonization to Dissemination into the Lungs.

Streptococcus pneumoniae is an asymptomatic colonizer of the nasopharynx, but it is also one of the most important bacterial pathogens of humans, causing a wide range of mild to life-threatening diseases. The basis of the pneumococcal transition from a commensal to a parasitic lifestyle is not fully understood. We hypothesize that exposure to host catecholamine stress hormones is important for this transition. In this study, we demonstrated that pneumococci preexposed to a hormone released during stress, norepinephrine (NE), have an increased capacity to translocate from the nasopharynx into the lungs compared to untreated pneumococci. Examination of NE-treated pneumococci revealed major alterations in metabolic profiles, cell associations, capsule synthesis, and cell size. By systemically mutating all 12 two-component and 1 orphan regulatory systems, we also identified a unique genetic regulatory circuit involved in pneumococcal recognition and responsiveness to human stress hormones. IMPORTANCE Microbes acquire unique lifestyles under different environmental conditions. Although this is a widespread occurrence, our knowledge of the importance of various host signals and their impact on microbial behavior is not clear despite the therapeutic value of this knowledge. We discovered that catecholamine stress hormones are the host signals that trigger the passage of Streptococcus pneumoniae from a commensal to a parasitic state. We identify that stress hormone treatment of this microbe leads to reductions in cell size and capsule synthesis and renders it more able to migrate from the nasopharynx into the lungs in a mouse model of infection. The microbe requires the TCS09 protein for the recognition and processing of stress hormone signals. Our work has particular clinical significance as catecholamines are abundant in upper respiratory fluids as well as being administered therapeutically to reduce inflammation in ventilated patients, which may explain why intubation in the critically ill is a recognized risk factor for the development of pneumococcal pneumonia.

Glutamate Dehydrogenase (GdhA) of Streptococcus pneumoniae Is Required for High Temperature Adaptation.

During its progression from the nasopharynx to other sterile and nonsterile niches of its human host, Streptococcus pneumoniae must cope with changes in temperature. We hypothesized that the temperature adaptation is an important facet of pneumococcal survival in the host. Here, we evaluated the effect of temperature on pneumococcus and studied the role of glutamate dehydrogenase (GdhA) in thermal adaptation associated with virulence and survival. Microarray analysis revealed a significant transcriptional response to changes in temperature, affecting the expression of 252 genes in total at 34°C and 40°C relative to at 37°C. One of the differentially regulated genes was gdhA, which is upregulated at 40°C and downregulated at 34°C relative to 37°C. Deletion of gdhA attenuated the growth, cell size, biofilm formation, pH survival, and biosynthesis of proteins associated with virulence in a temperature-dependent manner. Moreover, deletion of gdhA stimulated formate production irrespective of temperature fluctuation. Finally, ΔgdhA grown at 40°C was less virulent than other temperatures or the wild type at the same temperature in a Galleria mellonella infection model, suggesting that GdhA is required for pneumococcal virulence at elevated temperature.

Bruton's Tyrosine Kinase-Mediated Signaling in Myeloid Cells Is Required for Protective Innate Immunity During Pneumococcal Pneumonia.

Bruton's tyrosine kinase (Btk) is a cytoplasmic kinase expressed in B cells and myeloid cells. It is essential for B cell development and natural antibody-mediated host defense against bacteria in humans and mice, but little is known about the role of Btk in innate host defense in vivo. Previous studies have indicated that lack of (natural) antibodies is paramount for impaired host defense against Streptococcus (S.) pneumoniae in patients and mice with a deficiency in functional Btk. In the present study, we re-examined the role of Btk in B cells and myeloid cells during pneumococcal pneumonia and sepsis in mice. The antibacterial defense of Btk-/- mice was severely impaired during pneumococcal pneumosepsis and restoration of natural antibody production in Btk-/- mice by transgenic expression of Btk specifically in B cells did not suffice to protect against infection. Btk-/- mice with reinforced Btk expression in MhcII+ cells, including B cells, dendritic cells and macrophages, showed improved antibacterial defense as compared to Btk-/- mice. Bacterial outgrowth in Lysmcre-Btkfl/Y mice was unaltered despite a reduced capacity of Btk-deficient alveolar macrophages to respond to pneumococci. Mrp8cre-Btkfl/Y mice with a neutrophil specific paucity in Btk expression, however, demonstrated impaired antibacterial defense. Neutrophils of Mrp8cre-Btkfl/Y mice displayed reduced release of granule content after pulmonary installation of lipoteichoic acid, a gram-positive bacterial cell wall component relevant for pneumococci. Moreover, Btk deficient neutrophils showed impaired degranulation and phagocytosis upon incubation with pneumococci ex vivo. Taken together, the results of our study indicate that besides regulating B cell-mediated immunity, Btk is critical for regulation of myeloid cell-mediated, and particularly neutrophil-mediated, innate host defense against S. pneumoniae in vivo.

Critical Role of Zinc Transporter (ZIP8) in Myeloid Innate Immune Cell Function and the Host Response against Bacterial Pneumonia.

Zinc (Zn) is required for proper immune function and host defense. Zn homeostasis is tightly regulated by Zn transporters that coordinate biological processes through Zn mobilization. Zn deficiency is associated with increased susceptibility to bacterial infections, including Streptococcus pneumoniae, the most commonly identified cause of community-acquired pneumonia. Myeloid cells, including macrophages and dendritic cells (DCs), are at the front line of host defense against invading bacterial pathogens in the lung and play a critical role early on in shaping the immune response. Expression of the Zn transporter ZIP8 is rapidly induced following bacterial infection and regulates myeloid cell function in a Zn-dependent manner. To what extent ZIP8 is instrumental in myeloid cell function requires further study. Using a novel, myeloid-specific, Zip8 knockout model, we identified vital roles of ZIP8 in macrophage and DC function upon pneumococcal infection. Administration of S. pneumoniae into the lung resulted in increased inflammation, morbidity, and mortality in Zip8 knockout mice compared with wild-type counterparts. This was associated with increased numbers of myeloid cells, cytokine production, and cell death. In vitro analysis of macrophage and DC function revealed deficits in phagocytosis and increased cytokine production upon bacterial stimulation that was, in part, due to increased NF-κB signaling. Strikingly, alteration of myeloid cell function resulted in an imbalance of Th17/Th2 responses, which is potentially detrimental to host defense. These results (for the first time, to our knowledge) reveal a vital ZIP8- and Zn-mediated axis that alters the lung myeloid cell landscape and the host response against pneumococcus.

Inactivation of Transcriptional Regulator FabT Influences Colony Phase Variation of Streptococcus pneumoniae.

Streptococcus pneumoniae is an opportunistic pathogen that can alter its cell surface phenotype in response to the host environment. We demonstrated that the transcriptional regulator FabT is an indirect regulator of capsular polysaccharide, an important virulence factor of Streptococcus pneumoniae. Transcriptome analysis between the wild-type D39s and D39ΔfabT mutant strains unexpectedly identified a differentially expressed gene encoding a site-specific recombinase, PsrA. PsrA catalyzes the inversion of 3 homologous hsdS genes in a type I restriction-modification (RM) system SpnD39III locus and is responsible for the reversible switch of phase variation. Our study demonstrated that upregulation of PsrA in a D39ΔfabT mutant correlated with an increased ratio of transparent (T) phase variants. Inactivation of the invertase PsrA led to uniform opaque (O) variants. Direct quantification of allelic variants of hsdS derivatives and inversions of inverted repeats indicated that the recombinase PsrA fully catalyzes the inversion mediated by IR1 and IR3, and FabT mediated the recombination of the hsdS alleles in PsrA-dependent and PsrA-independent manners. In addition, compared to D39s, the ΔfabT mutant exhibited reduced nasopharyngeal colonization and was more resistant to phagocytosis and less adhesive to epithelial cells. These results indicated that phase variation in the ΔfabT mutant also affects other cell surface components involved in host interactions. IMPORTANCE Streptococcus pneumoniae is a major human pathogen, and its virulence factors and especially the capsular polysaccharide have been extensively studied. In addition to virulence components that are present on its cell surface that directly interact with the host, S. pneumoniae undergoes a spontaneous and reversible phase variation that allows survival in different host environments. This phase variation is manipulated by the recombination of allelic hsdS genes that encode the sequence recognition proteins of the type I RM system SpnD39III locus. The recombination of hsdS alleles is catalyzed by the DNA invertase PsrA. Interestingly, we found the opaque colony morphology can be reversed by inactivation of the transcriptional regulator FabT, which regulates fatty acid biosynthesis. Inactivation of FabT leads to a significant decrease in capsule production and systematic virulence, but these phase variations do not correlate with the capsule production. This phase variation is mediated via the upregulated invertase PsrA in the ΔfabT mutant. These results identify an unexpected link between the specific phase variations and FabT that strongly suggests an underlying mechanism regulating the DNA invertase PsrA.

Pneumolysin Is Responsible for Differential Gene Expression and Modifications in the Epigenetic Landscape of Primary Monocyte Derived Macrophages.

Epigenetic modifications regulate gene expression in the host response to a diverse range of pathogens. The extent and consequences of epigenetic modification during macrophage responses to Streptococcus pneumoniae, and the role of pneumolysin, a key Streptococcus pneumoniae virulence factor, in influencing these responses, are currently unknown. To investigate this, we infected human monocyte derived macrophages (MDMs) with Streptococcus pneumoniae and addressed whether pneumolysin altered the epigenetic landscape and the associated acute macrophage transcriptional response using a combined transcriptomic and proteomic approach. Transcriptomic analysis identified 503 genes that were differentially expressed in a pneumolysin-dependent manner in these samples. Pathway analysis highlighted the involvement of transcriptional responses to core innate responses to pneumococci including modules associated with metabolic pathways activated in response to infection, oxidative stress responses and NFκB, NOD-like receptor and TNF signalling pathways. Quantitative proteomic analysis confirmed pneumolysin-regulated protein expression, early after bacterial challenge, in representative transcriptional modules associated with innate immune responses. In parallel, quantitative mass spectrometry identified global changes in the relative abundance of histone post translational modifications (PTMs) upon pneumococcal challenge. We identified an increase in the relative abundance of H3K4me1, H4K16ac and a decrease in H3K9me2 and H3K79me2 in a PLY-dependent fashion. We confirmed that pneumolysin blunted early transcriptional responses involving TNF-α and IL-6 expression. Vorinostat, a histone deacetylase inhibitor, similarly downregulated TNF-α production, reprising the pattern observed with pneumolysin. In conclusion, widespread changes in the macrophage transcriptional response are regulated by pneumolysin and are associated with global changes in histone PTMs. Modulating histone PTMs can reverse pneumolysin-associated transcriptional changes influencing innate immune responses, suggesting that epigenetic modification by pneumolysin plays a role in dampening the innate responses to pneumococci.