Elise S. Hovingh

Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion

The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed.

Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface

Whooping cough, or pertussis, is a contagious disease of the respiratory tract that is re-emerging worldwide despite high vaccination coverage. The causative agent of this disease is the Gram-negative Bordetella pertussis. Knowledge on complement evasion strategies of this pathogen is limited. However, this is of great importance for future vaccine development as it has become apparent that a novel pertussis vaccine is needed. Here, we unravel the effect of Virulence associated gene 8 (Vag8) of B. pertussis on the human complement system at the molecular level. We show that both recombinant and endogenously secreted Vag8 inhibit complement deposition on the bacterial surface at the level of C4b. We reveal that Vag8 binding to human C1-inhibitor (C1-inh) interferes with the binding of C1-inh to C1s, C1r and MASP-2, resulting in the release of active proteases that subsequently cleave C2 and C4 away from the bacterial surface. We demonstrate that the depletion of these complement components in the bacterial surrounding and subsequent decreased deposition on B. pertussis leads to less complement-mediated bacterial killing. Vag8 is the first protein described that specifically prevents C1s, C1r and MASP-2 binding to C1-inh and thereby mediates complement consumption away from the bacterial surface. Unravelling the mechanism of this unique complement evasion strategy of B. pertussis is one of the first steps towards understanding the interactions between the first line of defense complement and B. pertussis.

Granzyme M cannot induce cell death via cleavage of mouse FADD.

To the Editor, Cytotoxic lymphocytes form the first line of defense against virus-infected and tumor cells, and can initiate target cell death via death receptor ligation or the granule exocytosis pathway [1]. In the latter pathway, cytotoxic lymphocytes deliver a set of structurally homologous serine proteases known as granzymes into the target cell, where they can activate intracellular cell death pathways [2]. Human granzyme M (hGrM) efficiently induces apoptosis in a wide variety of human tumor cells [3–6]. Recently, this induction of cell death has been shown to occur via hGrM-mediated cleavage of Fas-Associated protein with Death Domain (FADD) at Met, resulting in FADD self-association and initiation of the caspase cascade [6]. Replacement of FADD with a non-cleavable FADD mutant makes human tumor cells more resistant to hGrM-induced cell death, underlining the importance of this pathway [6]. Mice express the hGrM orthologue mouse GrM (mGrM). In mouse models, conflicting results have been obtained regarding the role of mGrM in tumor clearance. On the one hand, Pao et al. [7] found no effect of mGrM-deficiency on in vivo rejection of various tumor types, whilst Pegram et al. [8] noted that mGrM-deficient natural killer cells are significantly impaired in their ability to inhibit tumor growth. Interestingly, we have recently found that hGrM and mGrM exhibit only partially overlapping substrate specificities, and that both can efficiently induce apoptosis in human tumor cells, but not in various mouse tumor cells [4]. This suggests that key GrM substrates in human tumor cells are absent or cannot be cleaved in mouse tumor cells. In order to explain this apparent difference in GrM cytotoxicity towards human and mouse tumor cells, we investigated whether the recently unraveled pathway of hGrM-mediated cell death induction—via FADD cleavage—is intact in murine tumor cells. Although human FADD (hFADD) and mouse FADD (mFADD) display almost 70 % sequence homology, the GrM cleavage site at Met (P1 residue) in hFADD is not conserved in mFADD, which harbors a Val at this position (Fig. 1b). This suggests that GrM cannot cleave and activate FADD in a murine setting. In order to test this hypothesis, hGrM was incubated with lysates of the human HeLa cervix carcinoma and mouse C2C12 myoblast cell lines (Fig. 1b). We confirmed that GrM cleaves hFADD [6]. In mouse C2C12 lysate, however, no cleavage of mFADD could be observed (Fig. 1b), even though GrM was active in this lysate (data not shown). Similar results were obtained when isolated recombinant hFADD and mFADD were incubated with hGrM or mGrM (Fig. 1c). Whereas recombinant hFADD was efficiently processed by both hGrM and mGrM, mFADD was not cleaved by either (Fig. 1c). To restore GrM-mediated cleavage of mFADD, residues Val and Leu in mFADD were ‘humanized’ by substitution with the corresponding GrM cleavage site residues (P1 and P10) of hFADD, i.e. Met and Ser, respectively (Fig. 1c). In contrast to wild-type mFADD, this mFADD P1P10 mutant was cleaved by both mGrM and hGrM (Fig. 1c). Interestingly, hFADD seemed to be cleaved more efficiently by mGrM, which may be due to the presence of a Met at the P1 position, for which mGrM has a stronger preference [4]. Taken together, these data show that GrM cannot cleave FADD in mice because of a species-specific difference at S. A. H. de Poot K. W. Lai E. S. Hovingh N. Bovenschen (&) Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands e-mail: n.bovenschen@umcutrecht.nl

Emerging Bordetella pertussis Strains Induce Enhanced Signaling of Human Pattern Recognition Receptors TLR2, NOD2 and Secretion of IL-10 by Dendritic Cells

Vaccines against pertussis have been available for more than 60 years. Nonetheless, this highly contagious disease is reemerging even in countries with high vaccination coverage. Genetic changes of Bordetella pertussis over time have been suggested to contribute to the resurgence of pertussis, as these changes may favor escape from vaccine-induced immunity. Nonetheless, studies on the effects of these bacterial changes on the immune response are limited. Here, we characterize innate immune recognition and activation by a collection of genetically diverse B. pertussis strains isolated from Dutch pertussis patients before and after the introduction of the pertussis vaccines. For this purpose, we used HEK-Blue cells transfected with human pattern recognition receptors TLR2, TLR4, NOD2 and NOD1 as a high throughput system for screening innate immune recognition of more than 90 bacterial strains. Physiologically relevant human monocyte derived dendritic cells (moDC), purified from peripheral blood of healthy donors were also used. Findings indicate that, in addition to inducing TLR2 and TLR4 signaling, all B. pertussis strains activate the NOD-like receptor NOD2 but not NOD1. Furthermore, we observed a significant increase in TLR2 and NOD2, but not TLR4, activation by strains circulating after the introduction of pertussis vaccines. When using moDC, we observed that the recently circulating strains induced increased activation of these cells with a dominant IL-10 production. In addition, we observed an increased expression of surface markers including the regulatory molecule PD-L1. Expression of PD-L1 was decreased upon blocking TLR2. These in vitro findings suggest that emerging B. pertussis strains have evolved to dampen the vaccine-induced inflammatory response, which would benefit survival and transmission of this pathogen. Understanding how this disease has resurged in a highly vaccinated population is crucial for the design of improved vaccines against pertussis.

Detection of opsonizing antibodies directed against a recently circulating Bordetella pertussis strain in paired plasma samples from symptomatic and recovered pertussis patients

Correlates of protection (CoPs) against the highly contagious respiratory disease whooping cough, caused by Bordetella pertussis, remain elusive. Characterizing the antibody response to this pathogen is essential towards identifying potential CoPs. Here, we evaluate levels, avidity and functionality of B. pertussis-specific-antibodies from paired plasma samples derived from symptomatic and recovered pertussis patients, as well as controls. Natural infection is expected to induce protective immunity. IgG levels and avidity to nine B. pertussis antigens were determined using a novel multiplex panel. Furthermore,xa0opsonophagocytosis of a B. pertussis clinical isolate by neutrophils was measured. Findings indicate that following infection, B. pertussis-specific antibody levels of (ex-) pertussis patients waned, while the avidity of antibodies directed against the majority of studied antigens increased. Opsonophagocytosis indices decreased upon recovery, but remained higher than controls. Random forest analysis of all the data revealed that 28% of the opsonophagocytosis index variances could be explained by filamentous hemagglutinin- followed by pertussis toxin-specific antibodies. We propose to further explore which other B. pertussis-specific antibodies can better predict opsonophagocytosis. Moreover, other B. pertussis-specific antibody functions as well as the possible integration of these functions in combination with other immune cell properties should be evaluated towards the identification of CoPs against pertussis.

Virulence Associated Gene 8 of Enhances Contact System Activity by Inhibiting the Regulatory Function of Complement Regulator C1 Inhibitor

Bordetella pertussis is a Gram-negative bacterium and the causative agent of whooping cough. Whooping cough is currently re-emerging worldwide and, therefore, still poses a continuous global health threat. B. pertussis expresses several virulence factors that play a role in evading the human immune response. One of these virulence factors is virulence associated gene 8 (Vag8). Vag8 is a complement evasion molecule that mediates its effects by binding to the complement regulator C1 inhibitor (C1-INH). This regulatory protein is a fluid phase serine protease that controls proenzyme activation and enzyme activity of not only the complement system but also the contact system. Activation of the contact system results in the generation of bradykinin, a pro-inflammatory peptide. Here, the activation of the contact system by B. pertussis was explored. We demonstrate that recombinant as well as endogenous Vag8 enhanced contact system activity by binding C1-INH and attenuating its inhibitory function. Moreover, we show that B. pertussis itself is able to activate the contact system. This activation was dependent on Vag8 production as a Vag8 knockout B. pertussis strain was unable to activate the contact system. These findings show a previously overlooked interaction between the contact system and the respiratory pathogen B. pertussis. Activation of the contact system by B. pertussis may contribute to its pathogenicity and virulence.

Virulence associated gene 8 of Bordetella pertussis enhances contact system activity by inhibiting the regulatory function of C1 inhibitor

Bordetella pertussis is a Gram-negative bacterium and the causative agent of whooping cough. Whooping cough is currently re-emerging worldwide and, therefore, still poses a continuous global health threat. B. pertussis expresses several virulence factors that play a role in evading the human immune response. One of these virulence factors is virulence associated gene 8 (Vag8). Vag8 is a complement evasion molecule that mediates its effects by binding to the complement regulator C1 inhibitor (C1-INH). This regulatory protein is a fluid phase serine protease that controls proenzyme activation and enzyme activity of not only the complement system but also the contact system. Activation of the contact system results in the generation of bradykinin, a pro-inflammatory peptide. Here, the activation of the contact system by B. pertussis was explored. We demonstrate that recombinant as well as endogenous Vag8 enhanced contact system activity by binding C1-INH and attenuating its inhibitory function. Moreover, we show that B. pertussis itself is able to activate the contact system. This activation was dependent on Vag8 production as a Vag8 knockout B. pertussis strain was unable to activate the contact system. These findings show a previously overlooked interaction between the contact system and the respiratory pathogen B. pertussis. Activation of the contact system by B. pertussis may contribute to its pathogenicity and virulence.

Virulence Associated Gene 8 of Bordetella pertussis Enhances Contact System Activity by Inhibiting the Regulatory Function of Complement Regulator C1 Inhibitor.

Bordetella pertussis is a Gram-negative bacterium and the causative agent of whooping cough. Whooping cough is currently re-emerging worldwide and, therefore, still poses a continuous global health threat. B. pertussis expresses several virulence factors that play a role in evading the human immune response. One of these virulence factors is virulence associated gene 8 (Vag8). Vag8 is a complement evasion molecule that mediates its effects by binding to the complement regulator C1 inhibitor (C1-INH). This regulatory protein is a fluid phase serine protease that controls proenzyme activation and enzyme activity of not only the complement system but also the contact system. Activation of the contact system results in the generation of bradykinin, a pro-inflammatory peptide. Here, the activation of the contact system by B. pertussis was explored. We demonstrate that recombinant as well as endogenous Vag8 enhanced contact system activity by binding C1-INH and attenuating its inhibitory function. Moreover, we show that B. pertussis itself is able to activate the contact system. This activation was dependent on Vag8 production as a Vag8 knockout B. pertussis strain was unable to activate the contact system. These findings show a previously overlooked interaction between the contact system and the respiratory pathogen B. pertussis. Activation of the contact system by B. pertussis may contribute to its pathogenicity and virulence.

Bordetella pertussis pertactin knock-out strains reveal immunomodulatory properties of this virulence factor article

Whooping cough, caused by Bordetella pertussis, has resurged and presents a global health burden worldwide. B. pertussis strains unable to produce the acellular pertussis vaccine component pertactin (Prn), have been emerging and in some countries represent up to 95% of recent clinical isolates. Knowledge on the effect that Prn deficiency has on infection and immunity to B. pertussis is crucial for the development of new strategies to control this disease. Here, we characterized the effect of Prn production by B. pertussis on human and murine dendritic cell (DC) maturation as well as in a murine model for pertussis infection. We incubated human monocyte-derived DCs (moDCs) with multiple isogenic Prn knockout (Prn-KO) and corresponding parental B. pertussis strains constructed either in laboratory reference strains with a Tohama I background or in a recently circulating clinical isolate. Results indicate that, compared to the parental strains, Prn-KO strains induced an increased production of pro-inflammatory cytokines by moDCs. This pro-inflammatory phenotype was also observed upon stimulation of murine bone marrow-derived DCs. Moreover, RNA sequencing analysis of lungs from mice infected with B. pertussis Prn-KO revealed increased expression of genes involved in cell death. These in vitro and in vivo findings indicate that B. pertussis strains which do not produce Prn induce a stronger pro-inflammatory response and increased cell death upon infection, suggesting immunomodulatory properties for Prn.