Search

The obligate intracellular bacterium Chlamydia (C.) pneumoniae causes respiratory infections and is associated with vascular diseases. To elucidate how temperature and host cells used for propagation alter chlamydial virulence, C. pneumoniae CWL0129 (Cpn) was cultured at 35 or 37°C in two different cell lines and then applied to mice. These mice infected with differentially propagated chlamydiae showed differences in clinical score, body weight and inflammatory cytokines in the lung. Our study demonstrates that Cpn cultured at 37°C in hamster fibroblast BHK-21 are able to colonize the mouse lung faster and better, and induce stronger symptoms and cytokine induction than bacteria cultured at 35°C. The temperature-triggered virulence alteration could not be observed for Cpn propagated in HeLa cells and was independent of host cell protein synthesis. Transcriptome analysis did not reveal temperature-induced effects on chlamydial gene expression, suggesting that the observed virulence changes are regulated on a different, so far unknown level. Preculture close to the central body temperature of its warm-blooded human or murine host might 'prepare' Cpn for subsequent in vivo infection. Our identification of culture-dependent virulence alteration helps to establish an optimized mouse lung infection model for Cpn and provides the basis to further unravel the molecular mechanisms underlying chlamydial pathogenicity.

Hydrosalpinx is a pathological hallmark of tubal infertility associated with chlamydial infection. However, the mechanisms of hydrosalpinx remain unknown. Here, we report that complement factor 5 (C5) contributes significantly to chlamydial induction of hydrosalpinx. Mice lacking C5 (C5 −/− ) failed to develop any hydrosalpinx, while ∼42% of the corresponding wild-type mice (C5 +/+ ) did so following intravaginal infection with Chlamydia muridarum . Surprisingly, deficiency in C3 (C3 −/− ), an upstream component of the complement system, did not affect mouse susceptibility to chlamydial induction of hydrosalpinx. Interestingly, C5 activation was induced by chlamydial infection in oviducts of C3 −/− mice, explaining why the C3 −/− mice remained susceptible to chlamydial induction of hydrosalpinx. Similar levels of live chlamydial organisms were recovered from oviduct tissues of both C5 −/− and C5 +/+ mice, suggesting that C5 deficiency did not affect C. muridarum ascending infection. Furthermore, C5 −/− mice were still more resistant to hydrosalpinx induction than C5 +/+ mice, even when live C. muridarum organisms were directly delivered into the upper genital tract, both confirming the role of C5 in promoting hydrosalpinx and indicating that the C5-facilitated hydrosalpinx was not due to enhancement of ascending infection. The C5 −/− mice displayed significantly reduced lumenal inflammatory infiltration and cytokine production in oviduct tissue, suggesting that C5 may contribute to chlamydial induction of hydrosalpinx by enhancing inflammatory responses.

The intracellular gram-negative pathogen Chlamydia psittaci causes psittacosis in birds. Transmission of C. psittaci to humans can lead to life-threatening pneumonia with systemic dissemination [1, 2]. Infections of cattle and other domestic animals with non-avian strains can result in abortion, respiratory disorders, enteritis, and arthritis. Subclinical, protracted infections have impact on animal development and health [3–6]. All Chlamydia species including C. psittaci and other relevant human pathogens such as C. trachomatis and C. pneumoniae that cause genital, ocular, or respiratory infections, possess a virus-like biphasic developmental cycle characterized by extracellular, infectious “elementary bodies” and an intracellular, metabolically active form. These “reticulate bodies” reside within phagosome-like host cell inclusions [7, 8]. Toll-like receptors 2, 4, and Nod1 recognize chlamydial components. Neutrophils, macrophages, and T cells characterize the cellular response. Cytokines such as interleukin 1α/β (IL-1α/β), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 12 (IL-12), tumor necrosis factor α (TNF-α), granulocyte macrophage colony-stimulating factor (GM-CSF), and interferon γ (IFN-γ) orchestrate defense [9–11]. The humoral response ameliorates control of secondary infection with C. muridarum [12, 13]. Additionally, in genital reinfection caused by this mouse pathogen, Chlamydia-specific antiserum augments T cell immunity via Fc receptors on antigen-presenting cells (APCs), and enhances CD4+ T cell function [14, 15]. CD4+ and CD8+ T cells each are sufficient for effective defense against C. pneumoniae, with IFN-γ as key player [16]. The proteolytic complement cascade comprises essential defense against extracellular pathogens and links innate and adaptive immunity [17]. However, more recently there is evidence emerging that additionally, the complement system orchestrates adaptive immune responses and contributes to the control of intracellular pathogens [18–20]. Three main activation pathways lead to cleavage of complement factors C3 and C5 and release of the anaphylatoxins C3a and C5a. G protein-coupled receptors for C3a (C3aR) and C5a (C5aR, C5a1 receptor, CD88) mediate inflammation and play key roles in diseases including systemic lupus, arthritis, or colitis [17, 21, 22]. Although anaphylatoxin receptors are closely related, their functions overlap only partially; for example, in intestinal ischemia reperfusion injury C5a activates tissue-infiltrating neutrophils, whereas C3a limits neutrophil mobilization from bone marrow (BM) [23]. C5a serves as potent chemotaxin and activator of granulocytes and monocytes/macrophages [17]. In contrast, C3a only weakly affects these cells. The cleavage product C3b opsonizes pathogens promoting phagocytosis. Antigen-bound C3b (and C3d) augment antibody production and differentiation of B memory cells [24, 25]. Finally, the membrane attack complex downstream of C5 can lyse extracellular pathogens [26, 27]. Immune modulatory complement functions become increasingly relevant. APCs and activated T cells express anaphylatoxin receptors. Complement can affect cellular immunity by direct modulation of T cells or by enhancement of APC migration and antigen presentation [28–33]. There is little knowledge about complement during chlamydial infection: Elementary bodies activate complement in vitro, which reduces infectivity in cell culture [34–36]. Recently, our group has shown early, high, and long-lasting complement activation in C. psittaci mouse lung infection [19]. Experiments in C3−/− mice lacking all main effector functions revealed a protective role of complement in chlamydial infection. In contrast, the infection progressed similarly in wild type (WT), C5-deficient, and C5aR−/− animals, suggesting that biologically active C3 cleavage products play an important role. In the present study, we show a critical role of the C3aR in protection against intracellular C. psittaci. Whereas most WT mice survived C. psittaci infection, C3aR−/− mice had impaired bacterial clearance, prolonged inflammation, and a higher death rate. Moreover, C3aR−/− mice failed to raise C. psittaci-specific immunoglobulin M (IgM) and immunoglobulin G (IgG) and in comparison to WT mice developed weak B and C. psittaci-specific T cell responses in lung-draining lymph nodes (ldLNs). Our observations reveal a hitherto unknown role of the C3aR in adaptive immune responses towards an intracellular microorganism.

Chlamydia pneumoniae is associated with chronic inflammatory lung diseases like bronchial asthma and chronic obstructive pulmonary disease. The existence of a causal link between allergic airway disease and C. pneumoniae is controversial. A mouse model was used to address the question of whether preceding C. pneumoniae lung infection and recovery modifies the outcome of experimental allergic asthma after subsequent sensitization with house dust mite (HDM) allergen. After intranasal infection, BALB/c mice suffered from pneumonia characterized by an increased clinical score, reduction of body weight, histopathology, and a bacterial load in the lungs. After 4 weeks, when infection had almost resolved clinically, HDM allergen sensitization was performed for another 4 weeks. Subsequently, mice were subjected to a methacholine hyperresponsiveness test and sacrificed for further analyses. As expected, after 8 weeks, C. pneumoniae -specific antibodies were detectable only in infected mice and the titer was significantly higher in the C. pneumoniae /HDM allergen-treated group than in the C. pneumoniae /NaCl group. Intriguingly, airway hyperresponsiveness and eosinophilia in bronchoalveolar lavage fluid were significantly lower in the C. pneumoniae /HDM allergen-treated group than in the mock/HDM allergen-treated group. We did observe a relationship between experimental asthma and chlamydial infection. Our results demonstrate an influence of sensitization to HDM allergen on the development of a humoral antibacterial response. However, our model demonstrates no increase in the severity of experimental asthma to HDM allergen as a physiological allergen after clinically resolved severe chlamydial lung infection. Our results rather suggest that allergic airway disease and concomitant cellular changes in mice are decreased following C. pneumoniae lung infection in this setting.

INTRODUCTIONThe inflammasomes and the complement system are traditionally viewed as quintessential components of innate immunity required for the detection and elimination of pathogens. Assembly of the NLRP3 inflammasome in innate immune cells controls the maturation of interleukin (IL)–1β, a proinflammatory cytokine critical to host defense, whereas activation of the liver-derived complement key components C3 and C5 in serum leads to opsonization and removal of microbes and induction of the inflammatory reaction. Recent studies, however, have highlighted an unanticipated direct role for complement C3 also in human T cell immunity: The anaphylatoxin C3a receptor (C3aR) and the complement regulator CD46 (which binds C3b) are critical checkpoints in human T cell lineage commitment, and they control initiation and resolution of T helper 1 (TH1) responses in an autocrine fashion via T cell–derived and intracellularly activated C3. We explored a novel functional cross-talk of complement with the NLRP3 inflammasome within CD4+ T cells and determined how the cooperation between these two “classically” innate systems directly affects interferon-γ (IFN-γ) production by adaptive immune cells.RATIONALEGiven the critical role of intracellular C3 activation in human TH1 responses and the importance of C5 activation products in inflammation, we investigated whether human CD4+ T cells also harbor an “intracellular C5 activation system” and by what means this system may contribute to effector responses by using C5aR1 and C5aR2 agonists and antagonists, T cells from patients with cryopyrin-associated periodic syndromes (CAPS), and mouse models of infection and autoimmunity.RESULTSHuman CD4+ T cells expressed C5 and generated increased intracellular C5a upon T cell receptor activation and CD46 autocrine costimulation. Subsequent engagement of the intracellular C5aR1 by C5a induced the generation of reactive oxygen species (ROS) and the unexpected assembly of a functional NLRP3 inflammasome in CD4+ T cells, whereas the surface-expressed C5aR2 negatively controlled this process.NLRP3 inflammasome–dependent autocrine IL-1β secretion and activity were required for optimal IFN-γ production by T cells; consequently, dysregulation of NLRP3 function in these cells affected their normal effector responses. For example, mutated, constitutively active NLRP3 in T cells from patients with CAPS induced hyperactive TH1 responses that could be normalized with a NLRP3 inhibitor. The in vivo importance of a T cell–intrinsic NLRP3 inflammasome was further supported by the finding that IFN-γ production by Nlrp3–/– CD4+ T cells was significantly reduced during viral infections in mice and that diminished TH1 induction due to lack of NLRP3 function in a CD4+ T cell transfer model of colitis led to uncontrolled TH17 infiltration and/or expansion in the intestine and aggravated disease.CONCLUSIONOur results demonstrate that the regulated cross-talk between intracellularly activated complement components (the “complosome”) and the NLRP3 inflammasome is fundamental to human TH1 induction and regulation. The finding that established innate immune pathways are also operative in adaptive immune cells and orchestrate immunological responses contributes to our understanding of immunobiology and immune system evolution. In addition, the results suggest that the complement-NLRP3 axis in T cells represents a novel therapeutic target for the modulation of TH1 activity in autoimmunity and infection.

DivIVA is a conserved protein in Gram-positive bacteria that localizes at the poles and division sites, presumably through direct sensing of membrane curvature. DivIVA functions as a scaffold and is vital for septum site selection during vegetative growth and chromosome anchoring during sporulation. DivIVA deletion causes filamentous growth in Bacillus subtilis, whereas overexpression causes hyphal branching in Streptomyces coelicolor. We have determined the crystal structure of the N-terminal (Nt) domain of DivIVA, and show that it forms a parallel coiled-coil. It is capped with two unique crossed and intertwined loops, exposing hydrophobic and positively charged residues that we show here are essential for membrane binding. An intragenic suppressor introducing a positive charge restores membrane binding after mutating the hydrophobic residues. We propose that the hydrophobic residues insert into the membrane and that the positively charged residues bind to the membrane surface. A low-resolution crystal structure of the C-terminal (Ct) domain displays a curved tetramer made from two parallel coiled-coils. The Nt and Ct parts were then merged into a model of the full length, 30 nm long DivIVA protein.

With only 688 protein-coding genes, Mycoplasma pneumoniae is one of the smallest self-replicating organisms. These bacteria use glycolysis as the major pathway for ATP production by substrate-level phosphorylation, suggesting that this pathway must be optimized to high efficiency. In this study, we have investigated the interactions between glycolytic enzymes using the bacterial adenylate cyclase-based two-hybrid system. We demonstrate that most of the glycolytic enzymes perform self-interactions, suggesting that they form dimers or other oligomeric forms. In addition, enolase was identified as the central glycolytic enzyme of M. pneumoniae due to its ability to directly interact with all other glycolytic enzymes. Our results support the idea of the formation of a glycolytic complex in M. pneumoniae and we suggest that the formation of this complex might ensure higher fluxes through the glycolytic pathway than would be possible with isolated non-interacting enzymes.

Chlamydia trachomatis causes sexually transmitted diseases with infertility, pelvic inflammatory disease and neonatal pneumonia as complications. The duration of urogenital mouse models with the strict mouse pathogen C. muridarum addressing vaginal shedding, pathological changes of the upper genital tract or infertility is rather long. Moreover, vaginal C. trachomatis application usually does not lead to the complications feared in women. A fast-to-perform mouse model is urgently needed to analyze new antibiotics, vaccine candidates, immune responses (in gene knockout animals) or mutants of C. trachomatis. To complement the valuable urogenital model with a much faster and quantifiable screening method, we established an optimized lung infection model for the human intracellular bacterium C. trachomatis serovar D (and L2) in immunocompetent C57BL/6J mice. We demonstrated its usefulness by sensitive determination of antibiotic effects characterizing advantages and limitations achievable by early or delayed short tetracycline treatment and single-dose azithromycin application. Moreover, we achieved partial acquired protection in reinfection with serovar D indicating usability for vaccine studies, and showed a different course of disease in absence of complement factor C3. Sensitive monitoring parameters were survival rate, body weight, clinical score, bacterial load, histological score, the granulocyte marker myeloperoxidase, IFN-γ, TNF-α, MCP-1 and IL-6.

Virulent Streptococcus suis serotype 2 strains are invasive extracellular bacteria causing septicemia and meningitis in piglets and humans. One objective of this study was to elucidate the function of complement in innate immune defense against S. suis . Experimental infection of wild-type (WT) and C3 −/− mice demonstrated for the first time that the complement system protects naive mice against invasive mucosal S. suis infection. S. suis WT but not an unencapsulated mutant caused mortality associated with meningitis and other pathologies in C3 −/− mice. The capsule contributed also substantially to colonization of the upper respiratory tract. Experimental infection of C3 −/− mice with a suilysin mutant indicated that suilysin expression facilitated an early disease onset and the pathogenesis of meningitis. Flow cytometric analysis revealed C3 antigen deposition on the surface of ca. 40% of S. suis WT bacteria after opsonization with naive WT mouse serum, although to a significantly lower intensity than on the unencapsulated mutant. Ex vivo multiplication in murine WT and C3 −/− blood depended on capsule but not suilysin expression. Interestingly, S. suis invasion of inner organs was also detectable in C5aR −/− mice, suggesting that chemotaxis and activation of immune cells via the anaphylatoxin receptor C5aR is, in addition to opsonization, a further important function of the complement system in defense against mucosal S. suis infection. In conclusion, we unequivocally demonstrate here the importance of complement against mucosal S. suis serotype 2 infection and that the capsule of this pathogen is also involved in escape from complement-independent immunity.

With only 688 protein-coding genes, Mycoplasma pneumoniae is one of the smallest self-replicating organisms. These bacteria use glycolysis as the major pathway for ATP production by substrate-level phosphorylation, suggesting that this pathway must be optimized to high efficiency. In this study, we have investigated the interactions between glycolytic enzymes using the bacterial adenylate cyclase-based two-hybrid system. We demonstrate that most of the glycolytic enzymes perform self-interactions, suggesting that they form dimers or other oligomeric forms. In addition, enolase was identified as the central glycolytic enzyme of M. pneumoniae due to its ability to directly interact with all other glycolytic enzymes. Our results support the idea of the formation of a glycolytic complex in M. pneumoniae and we suggest that the formation of this complex might ensure higher fluxes through the glycolytic pathway than would be possible with isolated non-interacting enzymes.

The complement system modulates the intensity of innate and specific immunity. While it protects against infections by extracellular bacteria its role in infection with obligate intracellular bacteria, such as the avian and human pathogen Chlamydia (C.) psittaci, is still unknown. In the present study, knockout mice lacking C3 and thus all main complement effector functions were intranasally infected with C. psittaci strain DC15. Clinical parameters, lung histology, and cytokine levels were determined. A subset of infections was additionally performed with mice lacking C5 or C5a receptors. Complement activation occurred before symptoms of pneumonia appeared. Mice lacking C3 were ∼100 times more susceptible to the intracellular bacteria compared to wild-type mice, with all C3(-/-) mice succumbing to infection after day 9. At a low infective dose, C3(-/-) mice became severely ill after an even longer delay, the kinetics suggesting a so far unknown link of complement to the adaptive, protective immune response against chlamydiae. The lethal phenotype of C3(-/-) mice is not based on differences in the anti-chlamydial IgG response (which is slightly delayed) as demonstrated by serum transfer experiments. In addition, during the first week of infection, the absence of C3 was associated with partial protection characterized by reduced weight loss, better clinical score and lower bacterial burden, which might be explained by a different mechanism. Lack of complement functions downstream of C5 had little effect. This study demonstrates for the first time a strong and complex influence of complement effector functions, downstream of C3 and upstream of C5, on the outcome of an infection with intracellular bacteria, such as C. psittaci.

From 2017 to 2021, the Volkswagen Foundation went new ways for selecting small grantsin its funding initiative 'Experiment! - In Search of Bold Research Ideas'. In a partial randomization scheme, half of the grants have been selected by a jury as usual, the other half by a lottery after an initial quality check. Experiences, preliminary data, and results of accompanying research are shared.&nbsp