Andreas Nerlich

IFNs Modify the Proteome of Legionella-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid

Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.

The antimicrobial peptide LL-37 facilitates the formation of neutrophil extracellular traps

NETs (neutrophil extracellular traps) have been described as a fundamental innate immune defence mechanism. During formation of NETs, the nuclear membrane is disrupted by an as-yet unknown mechanism. In the present study we investigated the role of human cathelicidin LL-37 in nuclear membrane disruption and formation of NETs. Immunofluorescence microscopy revealed that 5 μM LL-37 significantly facilitated NET formation by primary human blood-derived neutrophils alone, in the presence of the classical chemical NET inducer PMA or in the presence of Staphylococcus aureus. Parallel assays with a random LL-37 fragment library indicated that the NET induction is mediated by the hydrophobic character of the peptide. The trans-localization of LL-37 towards the nucleus and the disruption of the nuclear membrane were visualized using confocal fluorescence microscopy. In conclusion, the present study demonstrates a novel role for LL-37 in the formation of NETs.

The FbaB-type fibronectin-binding protein of Streptococcus pyogenes promotes specific invasion into endothelial cells

Invasive serotype M3 Streptococcus pyogenes are among the most frequently isolated organisms from patients suffering from invasive streptococcal disease and have the potential to invade primary human endothelial cells (EC) via a rapid and efficient mechanism. FbaB protein, the fibronectin‐binding protein expressed by M3 S. pyogenes, was herein identified as a potent invasin for EC. By combining heterologous gene expression with allelic replacement, we demonstrate that FbaB is essential and sufficient to trigger EC invasion via a Rac1‐dependent phagocytosis‐like uptake. FbaB‐mediated uptake follows the classical endocytic pathway with lysosomal destination. FbaB is demonstrated to be a streptococcal invasin exhibiting EC tropism. FbaB thus initiates a process that may contribute to the deep tissue tropism and spread of invasive S. pyogenes isolates into the vascular EC lining.

SCM, a novel M-like protein from Streptococcus canis binds (Mini)-plasminogen with high affinity and facilitates bacterial transmigration

Streptococcus canis is an important zoonotic pathogen capable of causing serious invasive diseases in domestic animals and humans. In the present paper we report the binding of human plasminogen to S. canis and the recruitment of proteolytically active plasmin on its surface. The binding receptor for plasminogen was identified as a novel M-like protein designated SCM (S. canis M-like protein). SPR (surface plasmon resonance) analyses, radioactive dot-blot analyses and heterologous expression on the surface of Streptococcus gordonii confirmed the plasminogen-binding capability of SCM. The binding domain was located within the N-terminus of SCM, which specifically bound to the C-terminal part of plasminogen (mini-plasminogen) comprising kringle domain 5 and the catalytic domain. In the presence of urokinase, SCM mediated plasminogen activation on the bacterial surface that was inhibited by serine protease inhibitors and lysine amino acid analogues. Surface-bound plasmin effectively degraded purified fibrinogen as well as fibrin clots, resulting in the dissolution of fibrin thrombi. Electron microscopic illustration and time-lapse imaging demonstrated bacterial transmigration through fibrinous thrombi. The present study has led, for the first time, to the identification of SCM as a novel receptor for (mini)-plasminogen mediating the fibrinolytic activity of S. canis.

Invasion of Endothelial Cells by Tissue-invasive M3 Type Group A Streptococci Requires Src Kinase and Activation of Rac1 by a Phosphatidylinositol 3-Kinase-independent Mechanism

Streptococcus pyogenes can cause invasive diseases in humans, such as sepsis or necrotizing fasciitis. Among the various M serotypes of group A streptococci (GAS), M3 GAS lacks the major epithelial invasins SfbI/PrtF1 and M1 protein but has a high potential to cause invasive disease. We examined the uptake of M3 GAS into human endothelial cells and identified host signaling factors required to initiate streptococcal uptake. Bacterial uptake is accompanied by local F-actin accumulation and formation of membrane protrusions at the entry site. We found that Src kinases and Rac1 but not phos pha tidyl ino si tol 3-kinases (PI3Ks) are essential to mediate S. pyogenes internalization. Pharmacological inhibition of Src activity reduced bacterial uptake and abolished the formation of membrane protrusions and actin accumulation in the vicinity of adherent streptococci. We found that Src kinases are activated in a time-de pend ent manner in response to M3 GAS. We also demonstrated that PI3K is dispensable for internalization of M3 streptococci and the formation of F-actin accumulations at the entry site. Furthermore, Rac1 was activated in infected cells and accumulated with F-actin in a PI3K-independent manner at bacterial entry sites. Genetic interference with Rac1 function inhibited streptococcal internalization, demonstrating an essential role of Rac1 for the uptake process of streptococci into endothelial cells. In addition, we demonstrated for the first time accumulation of the actin nucleation complex Arp2/3 at the entry port of invading M3 streptococci.

Region specific and worldwide distribution of collagen-binding M proteins with PARF motifs among human pathogenic streptococcal isolates.

Some of the variety of Streptococcus pyogenes and Streptococcus dysgalactiae ssp. equisimilis (SDSE) M proteins act as collagen-binding adhesins that facilitate acute infection. Moreover, their potential to trigger collagen autoimmunity has been implicated in the pathogenesis of acute rheumatic fever and attributed to a collagen-binding motif called PARF (peptide associated with rheumatic fever). For the first time we determine the rate of clinical isolates with collagen-binding M proteins that use a PARF motif (A/T/E)XYLXX(L/F)N in a defined geographic region, Vellore in South India. In this region both, incidence of streptococcal infections and prevalence of acute rheumatic fever are high. M proteins with PARF motif conferred collagen-binding activity to 3.9% of 153 S. pyogenes and 10.6% of 255 SDSE clinical isolates from Vellore. The PARF motif occurred in three S. pyogenes and 22 SDSE M protein types. In one of the S. pyogenes and five of the SDSE M proteins that contained the motif, collagen-binding was impaired, due to influences of other parts of the M protein molecule. The accumulated data on the collagen binding activity of certain M protein types allowed a reanalysis of published worldwide emm-typing data with the aim to estimate the rates of isolates that bind collagen via PARF. The results indicate that M proteins, which bind collagen via a PARF motif, are epidemiologically relevant in human infections, not only in Vellore. It is imperative to include the most relevant collagen-binding M types in vaccines. But when designing M protein based vaccines it should be considered that collagen binding motifs within the vaccine antigen remain potential risk factors.

The CXC Chemokine-degrading Protease SpyCep of Streptococcus pyogenes Promotes Its Uptake into Endothelial Cells

Streptococcus pyogenes expresses the LPXTG motif-containing cell envelope serine protease SpyCep (also called ScpC, PrtS) that degrades and inactivates the major chemoattractant interleukin 8 (IL-8), thereby impairing host neutrophil recruitment. In this study, we identified a novel function of SpyCep: the ability to mediate uptake into primary human endothelial cells. SpyCep triggered its uptake into endothelial cells but not into human epithelial cells originating from pharynx or lung, indicating an endothelial cell-specific uptake mechanism. SpyCep mediated cellular invasion by an endosomal/lysosomal pathway distinct from the caveolae-mediated invasion pathway of S. pyogenes. Recombinant expression and purification of proteolytically active SpyCep and a series of subfragments allowed functional dissection of the domains responsible for endothelial cell invasion and IL-8 degradation. The N-terminal PR domain was sufficient to mediate endothelial cell invasion, whereas for IL-8-degrading activity, the protease domain and the flanking A domain were required. A polyclonal rabbit serum raised against the recombinant protease efficiently blocked the invasion-mediating activity of SpyCep but not its proteolytic function, further indicating that SpyCep-mediated internalization is independent from its enzymatic activity. SpyCep may thus specifically mediate its own uptake as secreted protein into human endothelial cells.

Streptococcus pneumoniae induces exocytosis of Weibel-Palade bodies in pulmonary endothelial cells.

Invasive pneumococcal infections due to Streptococcus pneumoniae lead to inflammatory infiltration of leucocytes into lung alveolus, meninges and to septic dissemination within the vascular system. The lung microvasculature is covered by pulmonary endothelial cells containing Weibel‐Palade bodies (WPB) releasing procoagulant von Willebrand factor (vWF) and other proteins in response to inflammatory stimuli. The influence of pathogenic pneumococci on secretion of WPB proteins is unknown. Here, we report that adherence of S. pneumoniae to primary human pulmonary microvascular endothelial cells (HPMEC) stimulates the WPB exocytosis and the secretion of vWF and interleukin 8 (IL‐8). Moreover, infection analyses performed with pneumococcal mutants deficient in the expression of cytotoxic pneumolysin demonstrated that, in addition to direct bacterial adherence, sublytic concentrations of pneumolysin stimulated vWF secretion. The release of vWF was induced after infection with pneumococci from both the apical and the basal cell surfaces, which implies a stimulation of WPB exocytosis in both directions: from inside the vasculature and also following invasive pneumococcal transmigration from pulmonary tissue into the bloodstream. In conclusion, this study demonstrates that the most relevant pulmonary pathogen S. pneumoniae induces release of proinflammatory and procoagulative components directly contributing to pathophysiological processes leading to fatal tissue injury during course of infection.

Clinical and Microbiologic Characteristics of Invasive Streptococcus pyogenes Infections in North and South India

ABSTRACT The lack of epidemiologic data on invasive Streptococcus pyogenes infections in many developing countries is concerning, as S. pyogenes infections are commonly endemic in these areas. Here we present the results of the first prospective surveillance study of invasive Streptococcus pyogenes infections in India. Fifty-four patients with invasive S. pyogenes infections were prospectively enrolled at two study sites, one in the north and one in the south of India. Sterile-site isolates were collected, and clinical information was documented using a standardized questionnaire. Available acute-phase sera were tested for their ability to inhibit superantigens produced by the patients own isolate using a cell-based neutralizing assay. The most common clinical presentations were bacteremia without focus (30%), pneumonia (28%), and cellulitis (17%). Only two cases of streptococcal toxic shock syndrome and no cases of necrotizing fasciitis were identified. Characterization of the isolates revealed great heterogeneity, with 32 different emm subtypes and 29 different superantigen gene profiles being represented among the 49 sterile-site isolates. Analyses of acute-phase sera showed that only 20% of the cases in the north cohort had superantigen-neutralizing activity in their sera, whereas 50% of the cases from the south site had neutralizing activity. The results demonstrate that there are important differences in both clinical presentation and strain characteristics between invasive S. pyogenes infections in India and invasive S. pyogenes infections in Western countries. The findings underscore the importance of epidemiologic studies on streptococcal infections in India and have direct implications for current vaccine developments.

cGAS-STING-TBK1-IRF3/7 induced interferon-β contributes to the clearing of non tuberculous mycobacterial infection in mice

Abstract Type I interferons (IFN-I), such as IFN-α and IFN-β are important messengers in the host response against bacterial infections. Knowledge about the role of IFN-I in infections by nontuberculous mycobacteria (NTM) is limited. Here we show that macrophages infected with pathogens of the Mycobacterium avium complex produced significantly lower amounts of IFN-β than macrophages infected with the opportunistic pathogen M. smegmatis. To dissect the molecular mechanisms of this phenomenon, we focused on the obligate pathogen Mycobacterium avium ssp paratuberculosis (MAP) and the opportunistic M. smegmatis. Viability of both bacteria was required for induction of IFN-β in macrophages. Both bacteria induced IFN-β via the cGAS-STING-TBK1-IRF3/7-pathway of IFN-β activation. Stronger phosphorylation of TBK1 and higher amounts of extracellular bacterial DNA in the macrophage cytosol were found in M. smegmatis infected macrophages than in MAP infected macrophages. After intraperitoneal infection of mice, a strong Ifnb induction by M. smegmatis correlated with clearance of the bacteria. In contrast, MAP only induced weak Ifnb expression which correlated with bacterial persistence and increased number of granulomas in the liver. In mice lacking the type I interferon receptor we observed improved survival of M. smegmatis while survival of MAP was similar to that in wildtype mice. On the other hand, treatment of MAP infected wildtype mice with the IFN-I inducer poly(I:C) or recombinant IFN-β impaired the survival of MAP. This indicates an essential role of IFN-I in clearing infections by MAP and M. smegmatis. The expression level of IFN-I is decisive for transient versus persistent NTM infection.