Mary C. Gray

The bacterial pathogen Neisseria gonorrhoeae is an urgent global health problem due to increasing numbers of infections, coupled with rampant antibiotic resistance. Vaccines against gonorrhea are being prioritized to combat drug-resistant N. gonorrhoeae. Meningococcal serogroup B vaccines such as 4CMenB are predicted by epidemiology studies to cross-protect individuals from natural infection with N. gonorrhoeae and elicit antibodies that cross-react with N. gonorrhoeae. Evaluation of vaccine candidates for gonorrhea requires a suite of assays for predicting efficacy in vitro and in animal models of infection, including the role of antibodies elicited by immunization. Here we present assays to evaluate antibody functionality after immunization: antibody binding to intact N. gonorrhoeae, serum bactericidal activity, and opsonophagocytic killing activity using primary human neutrophils (polymorphonuclear leukocytes). These assays were developed with purified antibodies against N. gonorrhoeae and used to evaluate serum from mice that were vaccinated with 4CMenB or given alum as a negative control. Results from these assays will help prioritize gonorrhea vaccine candidates for advanced preclinical to early clinical study and will contribute to identifying correlates and mechanisms of immune protection against N. gonorrhoeae .

Complement receptor 3 (CR3; CD11b/CD18; α _m β ₂ integrin) is a conserved phagocytic receptor. The active conformation of CR3 binds the iC3b fragment of complement C3 as well as many host and microbial ligands, leading to actin-dependent phagocytosis. There are conflicting reports about how CR3 engagement affects the fate of phagocytosed substrates. Using imaging flow cytometry, we confirmed that binding and internalization of iC3b-opsonized polystyrene beads by primary human neutrophils was CR3-dependent. iC3b-opsonized beads did not stimulate neutrophil reactive oxygen species (ROS), and most beads were found in primary granule-negative phagosomes. Similarly, Neisseria gonorrhoeae (Ngo) that does not express phase-variable Opa proteins suppresses neutrophil ROS and delays phagolysosome formation. Here, binding and internalization of Opa-deleted (Δopa) Ngo by adherent human neutrophils was inhibited using blocking antibodies against CR3 and by adding neutrophil inhibitory factor, which targets the CD11b I-domain. Neutrophils did not produce detectable amounts of C3 to opsonize Ngo. Conversely, overexpressing CD11b in HL-60 promyelocytes enhanced Δopa Ngo phagocytosis, which required CD11b I domain. Phagocytosis of Ngo was also inhibited in mouse neutrophils that were CD11b-deficient or treated with anti-CD11b. Phorbol ester treatment upregulated surface CR3 on neutrophils in suspension, enabling CR3-dependent phagocytosis of Δopa Ngo. Neutrophils exposed to Δopa Ngo had limited phosphorylation of Erk1/2, p38, and JNK. Neutrophil phagocytosis of unopsonized Mycobacterium smegmatis , which also resides in immature phagosomes, was CR3-dependent and did not elicit ROS. We suggest that CR3-mediated phagocytosis is a silent mode of entry into neutrophils, which is appropriated by diverse pathogens to subvert phagocytic killing.

Chinese hamster ovary (CHO) cells respond to pertussis toxin (PT) with a novel clustering pattern, which is dependent on biologically active PT. Since its description in 1983, this cellular response has been refined and used extensively for detection and quantification of PT activity, as well as anti-PT antibodies. There are limitations, however, in the use of this phenomenon as originally described. They are: (1) a subjective, observer-dependent scoring system; (2) the requirement for 16–24 h incubation in order for the response to be clearly detectable; and (3) apparent interference from non-toxin materials. To overcome these limitations, a number of alternative in vitro assays for PT, using CHO cells or other cell types, have been developed and are described elsewhere in this publication. In addressing the challenges associated with the CHO cell assay, we discovered that changes in the electrical impedance-based “normalized cell index” of PT-treated CHO cells obtained with the ACEA xCELLigence instrument enable objective detection/quantification of the PT-induced effect in as little as 3–4 h. To the best of our knowledge, the molecular basis for this intriguing response remains unknown. We present here electron microscopic (EM) images of control and PT-treated cells, which suggest some potential molecular mechanisms.

Infection with Neisseria gonorrhoeae (GC) is characterized by robust neutrophil influx that is insufficient to clear the bacteria. Sustained neutrophilic inflammation contributes to serious clinical sequelae that particularly affect women, including pelvic inflammatory disease and infertility. We established a 3-component system using GC, End1 polarized human endocervical cells, and primary human neutrophils to investigate neutrophil transepithelial migration following infection. Neutrophil migration across endocervical monolayers increased with the infectious dose and required GC-epithelial cell contact. Epithelial protein kinase C, cytosolic phospholipase A₂, 12R-lipoxygenase (LOX), and eLOX3 hepoxilin synthase were required for neutrophil transmigration to GC, and migration was abrogated by blocking the MRP2 efflux pump and by adding recombinant soluble epoxide hydrolase. These results are all consistent with epithelial cell production of the neutrophil chemoattractant hepoxilin A₃ (HXA₃). Neutrophil transmigration was also accompanied by increasing apical concentrations of leukotriene B₄ (LTB₄). Neutrophil 5-lipoxygenase and active BLT₁ receptor were required for apical LTB₄ and neutrophil migration. Our data support a model in which GC-endocervical cell contact infection stimulates HXA₃ production, driving neutrophil migration that is amplified by neutrophil-derived LTB₄. Therapeutic targeting of these pathways could limit inflammation and deleterious clinical sequelae in women with gonorrhea.

Bordetella pertussis is the primary causative agent of pertussis (whooping cough), which is a respiratory infection that leads to a violent cough and can be fatal in infants. There is a need to develop more effective vaccines because of the resurgence of cases of pertussis in the United States since the switch from the whole-cell pertussis vaccines (wP) to the acellular pertussis vaccines (aP; diphtheria-tetanus-acellular-pertussis vaccine/tetanus-diphtheria-pertussis vaccine).

ACT is an essential virulence factor of Bordetella pertussis and antibodies to ACT protect against B. pertussis infection in mice. The toxin is, therefore, a strong candidate antigen for addition to future acellular pertussis vaccines. In order to characterize the functionality of the immunologic response to ACT after infection, we have developed an assay for testing the ability of serum samples from subjects infected with B. pertussis to neutralize ACT-induced cytotoxicity in J774 macrophage cells. Baboons develop neutralizing anti-ACT antibodies following infection with B. pertussis, and all sera from baboons with positive anti-ACT IgG ELISA neutralized ACT cytotoxicity. The toxin neutralization assay (TNA) was positive in some baboon sera in which ELISA remained negative. Of serum samples obtained from humans diagnosed with pertussis by polymerase chain reaction, anti-ACT IgG ELISA was positive in 72% and TNA was positive in 83%. All samples positive for anti-ACT IgG ELISA were positive by TNA and none of the samples from humans without pertussis neutralized toxin activity. These findings indicate that antibodies to ACT generated following infection with B. pertussis consistently neutralize toxin-induced cytotoxicity, and that TNA can be used to improve understanding of the immunologic response to ACT after infection or vaccination.

Bordetella pertussis is a human pathogen that can infect the respiratory tract and cause the disease known as whooping cough. B. pertussis uses pertussis toxin (PT) and adenylate cyclase toxin (ACT) to kill and modulate host cells to allow the pathogen to survive and persist. B. pertussis encodes many uncharacterized transcription factors, and very little is known about their functions. RpoE is a sigma factor which, in other bacteria, responds to oxidative, heat, and other environmental stresses. RseA is a negative regulator of RpoE that sequesters the sigma factor to regulate gene expression based on conditions. In B. pertussis , deletion of the rseA gene results in high transcriptional activity of RpoE and large amounts of secretion of ACT. By comparing parental B. pertussis to an rseA gene deletion mutant (PM18), we sought to characterize the roles of RpoE in virulence and determine the regulon of genes controlled by RpoE. Despite high expression of ACT, the rseA mutant strain did not infect the murine airway as efficiently as the parental strain and PM18 was killed more readily when inside phagocytes. RNA sequencing analysis was performed and 263 genes were differentially regulated by RpoE, and surprisingly, the rseA mutant strain where RpoE activity was elevated expressed very little pertussis toxin. Western blots and proteomic analysis corroborated the inverse relationship of PT to ACT expression in the high-RpoE-activity rseA deletion strain. Our data suggest that RpoE can modulate PT and ACT expression indirectly through unidentified mechanisms in response to conditions.

Summary: Bordetella pertussis, the causative agent of whooping cough, secretes and releases adenylate cyclase toxin (ACT), which is a protein bacterial toxin that targets host cells and disarms immune defenses. ACT binds filamentous haemagglutinin (FHA), a surface‐displayed adhesin, and until now, the consequences of this interaction were unknown. A B. bronchiseptica mutant lacking ACT produced more biofilm than the parental strain; leading Irie et al. to propose the ACT‐FHA interaction could be responsible for biofilm inhibition. Here we characterize the physical interaction of ACT with FHA and provide evidence linking that interaction to inhibition of biofilm in vitro. Exogenous ACT inhibits biofilm formation in a concentration‐dependent manner and the N‐terminal catalytic domain of ACT (AC domain) is necessary and sufficient for this inhibitory effect. AC Domain interacts with the C‐terminal segment of FHA with ∼650 nM affinity. ACT does not inhibit biofilm formation by Bordetella lacking the mature C‐terminal domain (MCD), suggesting the direct interaction between AC domain and the MCD is required for the inhibitory effect. Additionally, AC domain disrupts preformed biofilm on abiotic surfaces. The demonstrated inhibition of biofilm formation by a host‐directed protein bacterial toxin represents a novel regulatory mechanism and identifies an unprecedented role for ACT.

Clostridium difficile toxins A and B (TcdA and TcdB), considered to be essential for C. difficile infection, affect the morphology of several cell types with different potencies and timing. However, morphological changes over various time scales are poorly characterized. The toxins’ glucosyltransferase domains are critical to their deleterious effects, and cell responses to glucosyltransferase-independent activities are incompletely understood. By tracking morphological changes of multiple cell types to C. difficile toxins with high temporal resolution, cellular responses to TcdA, TcdB, and a glucosyltransferase-deficient TcdB (gdTcdB) are elucidated. Human umbilical vein endothelial cells, J774 macrophage-like cells, and four epithelial cell lines (HCT8, T84, CHO, and immortalized mouse cecal epithelial cells) were treated with TcdA, TcdB, gdTcdB. Impedance across cell cultures was measured to track changes in cell morphology. Metrics from impedance data, developed to quantify rapid and long-lasting responses, produced standard curves with wide dynamic ranges that defined cell line sensitivities. Except for T84 cells, all cell lines were most sensitive to TcdB. J774 macrophages stretched and increased in size in response to TcdA and TcdB but not gdTcdB. High concentrations of TcdB and gdTcdB (>10 ng/ml) greatly reduced macrophage viability. In HCT8 cells, gdTcdB did not induce a rapid cytopathic effect, yet it delayed TcdA and TcdB’s rapid effects. gdTcdB did not clearly delay TcdA or TcdB’s toxin-induced effects on macrophages. Epithelial and endothelial cells have similar responses to toxins yet differ in timing and degree. Relative potencies of TcdA and TcdB in mouse epithelial cells in vitro do not correlate with potencies in vivo. TcdB requires glucosyltransferase activity to cause macrophages to spread, but cell death from high TcdB concentrations is glucosyltransferase-independent. Competition experiments with gdTcdB in epithelial cells confirm common TcdA and TcdB mechanisms, yet different responses of macrophages to TcdA and TcdB suggest different, additional mechanisms or targets in these cells. This first-time, precise quantification of the response of multiple cell lines to TcdA and TcdB provides a comparative framework for delineating the roles of different cell types and toxin-host interactions.