Anders P. Hakansson

In Vivo Activation of Dendritic Cells and T Cells during Salmonella enterica Serovar Typhimurium Infection

ABSTRACT The present study was initiated to gain insight into the interaction between splenic dendritic cells (DC) and Salmonella enterica serovar Typhimurium in vivo. Splenic phagocytic cell populations associated with green fluorescent protein (GFP)-expressing bacteria and the bacterium-specific T-cell response were evaluated in mice given S. enterica serovar Typhimurium expressing GFP and ovalbumin. Flow cytometry analysis revealed that GFP-positive splenic DC (CD11c+ major histocompatibility complex class II-positive [MHC-II+] cells) were present following bacterial administration, and confocal microscopy showed that GFP-expressing bacteria were contained within CD11c+MHC-II+ splenocytes. Furthermore, splenic DC and T cells were activated following Salmonella infection. This was shown by increased surface expression of CD86 and CD40 on CD11c+ MHC-II+ cells and increased CD44 and CD69 expression on CD4+ and CD8+ T cells.Salmonella-specific gamma interferon (IFN-γ)-producing cells in both of these T-cell subsets, as well as cytolytic effector cells, were also generated in mice given live bacteria. The frequency of Salmonella-specific CD4+ T cells producing IFN-γ was greater than that of specific CD8+ T cells producing IFN-γ in the same infected animal. This supports the argument that the predominant source of IFN-γ production by cells of the specific immune response is CD4+ T cells. Finally, DC that phagocytosed live or heat-killed Salmonella in vitro primed bacterium-specific IFN-γ-producing CD4+ and CD8+ T cells as well as cytolytic effector cells following administration into naı̈ve mice. Together these data suggest that DC are involved in priming naı̈ve T cells toSalmonella in vivo.

Interkingdom Signaling Induces Streptococcus pneumoniae Biofilm Dispersion and Transition from Asymptomatic Colonization to Disease

ABSTRACT Streptococcus pneumoniae is a common human nasopharyngeal commensal colonizing 10% to 40% of healthy individuals, depending on age. Despite a low invasive disease rate, widespread carriage ensures that infection occurs often enough to make S. pneumoniae a leading bacterial cause of respiratory disease worldwide. However, the mechanisms behind transition from asymptomatic colonization to dissemination and disease in otherwise sterile sites remain poorly understood but are epidemiologically strongly linked to infection with respiratory viruses. In this report, we show that infection with influenza A virus and treatment with the resulting host signals (febrile-range temperatures, norepinephrine, extracytoplasmic ATP, and increased nutrient availability) induce the release of bacteria from biofilms in a newly developed biofilm model on live epithelial cells both in vitro and during in vivo colonization. These dispersed bacteria have distinct phenotypic properties different from those of both biofilm and broth-grown, planktonic bacteria, with the dispersed population showing differential virulence gene expression characteristics resulting in a significantly increased ability to disseminate and cause infection of otherwise sterile sites, such as the middle ear, lungs, and bloodstream. The results offer novel and important insights into the role of interkingdom signaling between microbe and host during biofilm dispersion and transition to acute disease. IMPORTANCE This report addresses the mechanisms involved in transition from pneumococcal asymptomatic colonization to disease. In this study, we determined that changes in the nasopharyngeal environment result in the release of bacteria from colonizing biofilms with a gene expression and virulence phenotype different not only from that of colonizing biofilm bacteria but also from that of the broth-grown planktonic bacteria commonly used for pathogenesis studies. The work importantly also identifies specific host factors responsible for the release of bacteria and their changed phenotype. We show that these interkingdom signals are recognized by bacteria and are induced by influenza virus infection, which is epidemiologically strongly associated with transition to secondary pneumococcal disease. As virus infection is a common inducer of transition to disease among species occupying the nasopharynx, the results of this study may provide a basis for better understanding of the signals involved in the transition from colonization to disease in the human nasopharynx. This report addresses the mechanisms involved in transition from pneumococcal asymptomatic colonization to disease. In this study, we determined that changes in the nasopharyngeal environment result in the release of bacteria from colonizing biofilms with a gene expression and virulence phenotype different not only from that of colonizing biofilm bacteria but also from that of the broth-grown planktonic bacteria commonly used for pathogenesis studies. The work importantly also identifies specific host factors responsible for the release of bacteria and their changed phenotype. We show that these interkingdom signals are recognized by bacteria and are induced by influenza virus infection, which is epidemiologically strongly associated with transition to secondary pneumococcal disease. As virus infection is a common inducer of transition to disease among species occupying the nasopharynx, the results of this study may provide a basis for better understanding of the signals involved in the transition from colonization to disease in the human nasopharynx.

High Levels of Genetic Recombination during Nasopharyngeal Carriage and Biofilm Formation in Streptococcus pneumoniae

ABSTRACT Transformation of genetic material between bacteria was first observed in the 1920s using Streptococcus pneumoniae as a model organism. Since then, the mechanism of competence induction and transformation has been well characterized, mainly using planktonic bacteria or septic infection models. However, epidemiological evidence suggests that genetic exchange occurs primarily during pneumococcal nasopharyngeal carriage, which we have recently shown is associated with biofilm growth, and is associated with cocolonization with multiple strains. However, no studies to date have comprehensively investigated genetic exchange during cocolonization in vitro and in vivo or the role of the nasopharyngeal environment in these processes. In this study, we show that genetic exchange during dual-strain carriage in vivo is extremely efficient (10−2) and approximately 10,000,000-fold higher than that measured during septic infection (10−9). This high transformation efficiency was associated with environmental conditions exclusive to the nasopharynx, including the lower temperature of the nasopharynx (32 to 34°C), limited nutrient availability, and interactions with epithelial cells, which were modeled in a novel biofilm model in vitro that showed similarly high transformation efficiencies. The nasopharyngeal environmental factors, combined, were critical for biofilm formation and induced constitutive upregulation of competence genes and downregulation of capsule that promoted transformation. In addition, we show that dual-strain carriage in vivo and biofilms formed in vitro can be transformed during colonization to increase their pneumococcal fitness and also, importantly, that bacteria with lower colonization ability can be protected by strains with higher colonization efficiency, a process unrelated to genetic exchange. IMPORTANCE Although genetic exchange between pneumococcal strains is known to occur primarily during colonization of the nasopharynx and colonization is associated with biofilm growth, this is the first study to comprehensively investigate transformation in this environment and to analyze the role of environmental and bacterial factors in this process. We show that transformation efficiency during cocolonization by multiple strains is very high (around 10−2). Furthermore, we provide novel evidence that specific aspects of the nasopharyngeal environment, including lower temperature, limited nutrient availability, and epithelial cell interaction, are critical for optimal biofilm formation and transformation efficiency and result in bacterial protein expression changes that promote transformation and fitness of colonization-deficient strains. The results suggest that cocolonization in biofilm communities may have important clinical consequences by facilitating the spread of antibiotic resistance and enabling serotype switching and vaccine escape as well as protecting and retaining poorly colonizing strains in the pneumococcal strain pool. Although genetic exchange between pneumococcal strains is known to occur primarily during colonization of the nasopharynx and colonization is associated with biofilm growth, this is the first study to comprehensively investigate transformation in this environment and to analyze the role of environmental and bacterial factors in this process. We show that transformation efficiency during cocolonization by multiple strains is very high (around 10−2). Furthermore, we provide novel evidence that specific aspects of the nasopharyngeal environment, including lower temperature, limited nutrient availability, and epithelial cell interaction, are critical for optimal biofilm formation and transformation efficiency and result in bacterial protein expression changes that promote transformation and fitness of colonization-deficient strains. The results suggest that cocolonization in biofilm communities may have important clinical consequences by facilitating the spread of antibiotic resistance and enabling serotype switching and vaccine escape as well as protecting and retaining poorly colonizing strains in the pneumococcal strain pool.

Characterization of binding of human lactoferrin to pneumococcal surface protein A

ABSTRACT Human lactoferrin is an iron-binding glycoprotein that is particularly prominent in exocrine secretions and leukocytes and is also found in serum, especially during inflammation. It is able to sequester iron from microbes and has immunomodulatory functions, including inhibition of both complement activation and cytokine production. This study used mutants lacking pneumococcal surface protein A (PspA) and PspC to demonstrate that the binding of human lactoferrin to the surface of Streptococcus pneumoniae was entirely dependent on PspA. Lactoferrin bound both family 1 and family 2 PspAs. Binding of lactoferrin to PspA was shown by surface colocalization with PspA and was verified by the lack of binding to PspA-negative mutants. Lactoferrin was expressed on the body of the cells but was largely absent from the poles. PspC showed exactly the same distribution on the pneumococcal surface as PspA but did not bind lactoferrin. PspAs binding site for lactoferrin was mapped using recombinant fragments of PspA of families 1 and 2. Binding of human lactoferrin was detected primarily in the C-terminal half of the α-helical domain of PspA (amino acids 167 to 288 of PspA/Rx1), with no binding to the N-terminal 115 amino acids in either strain. The interaction was highly specific. As observed previously, bovine lactoferrin bound poorly to PspA. Human transferrin did not bind PspA at all. The binding of lactoferrin to S. pneumoniae might provide a way for the bacteria to interfere with host immune functions or to aid in the acquisition of iron at the site of infection.

Pneumococcal Interactions with Epithelial Cells Are Crucial for Optimal Biofilm Formation and Colonization In Vitro and In Vivo

ABSTRACT The human nasopharynx is the main reservoir for Streptococcus pneumoniae (the pneumococcus) and the source for both horizontal spread and transition to infection. Some clinical evidence indicates that nasopharyngeal carriage is harder to eradicate with antibiotics than is pneumococcal invasive disease, which may suggest that colonizing pneumococci exist in biofilm communities that are more resistant to antibiotics. While pneumococcal biofilms have been observed during symptomatic infection, their role in colonization and the role of host factors in this process have been less studied. Here, we show for the first time that pneumococci form highly structured biofilm communities during colonization of the murine nasopharynx that display increased antibiotic resistance. Furthermore, pneumococcal biofilms grown on respiratory epithelial cells exhibited phenotypes similar to those observed during colonization in vivo, whereas abiotic surfaces produced less ordered and more antibiotic-sensitive biofilms. The importance of bacterial-epithelial cell interactions during biofilm formation was shown using both clinical strains with variable colonization efficacies and pneumococcal mutants with impaired colonization characteristics in vivo. In both cases, the ability of strains to form biofilms on epithelial cells directly correlated with their ability to colonize the nasopharynx in vivo, with colonization-deficient strains forming less structured and more antibiotic-sensitive biofilms on epithelial cells, an association that was lost when grown on abiotic surfaces. Thus, these studies emphasize the importance of host-bacterial interactions in pneumococcal biofilm formation and provide the first experimental data to explain the high resistance of pneumococcal colonization to eradication by antibiotics.

Apoptosis-Like Death in Bacteria Induced by HAMLET, a Human Milk Lipid-Protein Complex

Background Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. Methodology/Principal Findings We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death. Conclusions/Significance Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells.

Oleic acid is a key cytotoxic component of HAMLET-like complexes

Abstract HAMLET is a complex of α-lactalbumin (α-LA) with oleic acid (OA) that selectively kills tumor cells and Streptococcus pneumoniae. To assess the contribution of the proteinaceous component to cytotoxicity of HAMLET, OA complexes with proteins structurally and functionally distinct from α-LA were prepared. Similar to HAMLET, the OA complexes with bovine β-lactoglobulin (bLG) and pike parvalbumin (pPA) (bLG-OA-45 and pPA-OA-45, respectively) induced S. pneumoniae D39 cell death. The activation mechanisms of S. pneumoniae death for these complexes were analogous to those for HAMLET, and the cytotoxicity of the complexes increased with OA content in the preparations. The half-maximal inhibitory concentration for HEp-2 cells linearly decreased with rise in OA content in the preparations, and OA concentration in the preparations causing HEp-2 cell death was close to the cytotoxicity of OA alone. Hence, the cytotoxic action of these complexes against HEp-2 cells is induced mostly by OA. Thermal stabilization of bLG upon association with OA implies that cytotoxicity of bLG-OA-45 complex cannot be ascribed to molten globule-like conformation of the protein component. Overall, the proteinaceous component of HAMLET-like complexes studied is not a prerequisite for their activity; the cytotoxicity of these complexes is mostly due to the action of OA.

Mg 2+ signalling defines the group A streptococcal CsrRS (CovRS) regulon

CsrRS (or CovRS) is a two‐component system implicated in the control of multiple virulence determinants in the important human pathogen, group A Streptococcus (GAS). Earlier studies suggested that extracellular Mg2+ signalled through the presumed sensor histidine kinase, CsrS. We now confirm those findings, as complementation of a csrS mutant restored Mg2+‐dependent gene regulation. Moreover, we present strong evidence that Mg2+ signals through CsrS to regulate an extensive and previously undefined repertoire of GAS genes. The effect of Mg2+ on regulation of global gene expression was evaluated using genomic microarrays in an M‐type 3 strain of GAS and in an isogenic csrS mutant. Unexpectedly, of the 72 genes identified in the Mg2+‐stimulated CsrRS regulon, 42 were absent from the CsrR regulon (the latter being defined by comparison of wild‐type and CsrR mutant transcriptomes at low Mg2+). We observed CsrS‐dependent regulation of 72 of the 73 genes whose expression changed in response to elevated extracellular Mg2+ in wild‐type bacteria, a result that identifies CsrS as the principal, if not exclusive, sensor for extracellular Mg2+ in GAS. To our knowledge, this study is the first to characterize global gene regulation by a GAS two‐component system in response to a specific environmental stimulus.

Morphological and functional in vitro and in vivo characterization of the mouse corpus cavernosum.

In normal mice, the distribution of adrenergic, cholinergic, some peptidergic, and neuronal nitric oxide synthase (nNOS)‐containing nerves were investigated. Functional in vitro correlates were obtained. An in vivo model was developed in which erectile haemodynamics in response to drugs or nerve‐stimulation were studied. Immunoreactivities for vesicular acetylcholine transporter protein (VAChT), nNOS‐, and vasoactive intestinal polypeptide (VIP), co‐existed in nerve fibres and terminal varicosities. Immunoreactivities for neuropeptide Y (NPY) and tyrosine hydroxylase (TH) were found in the same nerve structures. Chemical sympathectomy abolished TH‐ and NPY‐IR nerve structures in cavernous smooth muscle bundles. The distribution of calcitonin gene‐related peptide (CGRP)‐, nNOS‐, VAChT‐ and VIP‐IR nerve structures was unchanged. In endothelial cells of the central and helicine arteries, veins and venules, intense immunoreactivity for endothelial NOS (eNOS) was observed. No distinct eNOS‐IR cells were found lining the cavernous sinusoids. In vitro, nerve‐induced relaxations were verified, and endothelial NO/cyclic GMP‐mediated relaxant responses were established. VIP and CGRP had small relaxant effects. A functioning adenylate cyclase/cyclic AMP pathway was confirmed. Neuronal excitatory responses were abolished by prazosin, or forskolin. VIP and CGRP counteracted contractions, whereas NPY and scopolamine enhanced excitatory responses. In vivo, erectile responses were significantly attenuated by L‐NAME (50u2003mgu2003kg−1) and facilitated by sildenafil (200u2003μgu2003kg−1). It is concluded that the mouse is a suitable model for studies of erectile mechanisms in vitro and in vivo.

Extracellular group A Streptococcus induces keratinocyte apoptosis by dysregulating calcium signalling

Group A Streptococcus (GAS) colonizes the oropharynx and damaged skin. To cause local infection or severe invasive syndromes the bacteria must gain access into deeper tissues. Host cell death may facilitate this process. GAS internalization has been identified to induce apoptosis. We now report an alternate mechanism of GAS‐mediated apoptosis of primary human keratinocytes, initiated by extracellular GAS and involving dysregulation of intracellular calcium to produce endoplasmic reticulum stress. Two bacterial virulence factors are required for effective induction of apoptosis by extracellular GAS: (i) hyaluronic acid capsule that inhibits bacterial internalization and (ii) secreted cytolysin, streptolysin O (SLO), that forms transmembrane pores that permit extracellular calcium influx into the cytosol. Induction of keratinocyte apoptosis by wild‐type GAS was accompanied by cell detachment and loss of epithelial integrity, a phenomenon not observed with GAS deficient in capsule or SLO. We propose that cell signalling initiated by extracellular GAS compromises the epithelial barrier by inducing premature keratinocyte differentiation and apoptosis, thereby facilitating GAS invasion of deeper tissues.