Biology subjects

Criss, A. K.

Publications and source records attributed to Criss, A. K..

13 recordsLinked to original sources

bioRxiv2026

Pre-clinical efficacy of a C4BP hexameric IgG Fc fusion protein against Neisseria gonorrhoeae

Gonorrhea is the second most common bacterial sexually transmitted infection and affects about 80 million people worldwide annually. The causative agent, Neisseria gonorrhoeae, has become resistant to almost every antibiotic used for its treatment. There is no licensed vaccine against gonorrhea. Therefore, there is an urgent need to develop novel prevention and treatment strategies to curb the spread of gonorrhea. The gonococcus has evolved several mechanisms to evade complement, a key arm of immune defenses against this pathogen, including binding of the human complement inhibitors Factor H (FH) and C4b-binding protein (C4BP). We previously showed that chimeric molecules fusing the gonococcal binding domains of FH and C4BP to IgG Fc and IgM Fc, respectively, mediate complement-dependent killing of gonococci in vitro and attenuate gonococcal colonization of mouse vaginas when administered topically. Here, we fused C4BP domains 1 and 2, which contain the gonococcal binding region, to IgG Fc bearing the IgM tail-piece to facilitate Fc hexamerization. This molecule, called C4BP-Hexa IgG Fc, showed [~]650-fold greater complement-dependent bactericidal activity on a molar basis than monomeric C4BP-IgG1 Fc. C4BP-Hexa IgG Fc enhanced association with and uptake by human neutrophils in a complement-independent manner. Despite off-target complement activation in solution, C4BP-Hexa IgG Fc reduced both the duration and the bacterial burden of gonococcal vaginal colonization in human FH and C4BP transgenic mice when administered intravaginally daily. In conclusion, we show proof-of-concept of the efficacy of a hexameric C4BP IgG Fc fusion molecule against N. gonorrhoeae, which could aid in the fight against this multidrug-resistant pathogen.

immunology↗

bioRxiv2026

C4BP occludes the non-opsonic interaction of Neisseria gonorrhoeae with human neutrophil CEACAMs

Neisseria gonorrhoeae (Gc) causes the sexually transmitted infection gonorrhea, an urgent public health concern. Gc infection elicits a robust neutrophil response and serum leakage, but Gc has developed specialized defenses to evade both complement and neutrophils. We recently reported that the classical complement pathway inhibitor C4b-binding protein (C4BP) binds to Gc and reduces phagocytic killing by neutrophils in a complement-independent manner. Here, we used a Chinese hamster ovary (CHO) expression system and engineered C4BP constructs to define the underlying molecular mechanisms. C4BP inhibited interactions between opacity protein (Opa)-expressing Gc and carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), receptors that drive non-opsonic phagocytosis of Gc by neutrophils. The degree of C4BP-mediated inhibition varied among CEACAMs. By using wild-type and chimeric CEACAMs, we found C4BP was more inhibitory towards the granulocyte-restricted CEACAM3 than the ubiquitously expressed CEACAM1, which we ascribed to CEACAM3s shorter extracellular domain. C4BP also inhibited the association between Opa-expressing Gc and the GPI-anchored CEACAM6. Molecules containing C4BP domains 1 and 2 fused to IgM (C4BP-IgM) or to a hexameric IgG Fc construct (C4BP-Hexa-IgG), proteins similar in diameter and degree of multimerization to native C4BP, inhibited the association of Opa-expressing Gc with CEACAM3-CHO cells to the same degree as C4BP, while C4BP domains 1 and 2 fused to dimeric Fc (C4BP-IgG) did not. C4BP-IgM, but not C4BP-IgG bearing mutations to abrogate Fc gamma receptor interactions, blocked Opa-mediated phagocytosis by primary human neutrophils. These results support a model in which C4BP occludes Opa-CEACAM interactions, which protects Gc from phagocytic killing by neutrophils.

microbiology↗

bioRxiv2025

The L-lactate dehydrogenase LldD contributes to oxidative stress resistance, survival from neutrophils, and host colonization in Neisseria gonorrhoeae

Metabolic adaptation to the host environment is a key determinant of bacterial pathogenesis, enabling both colonization and invasive disease. This is particularly true for Neisseria gonorrhoeae (Gc), the causative agent of gonorrhea, which lacks effector-injecting secretion systems or toxins. Gc infection triggers a rapid influx of neutrophils (PMNs) that typically kill bacteria through multiple mechanisms, including a potent oxidative burst. Despite this, Gc exhibits remarkable resistance to reactive oxygen species and readily replicates in the presence of PMNs, which is in part due to the consumption of PMN-derived lactate. Previous studies demonstrated that the lactate permease, LctP, is required for oxidative stress resistance in Gc and host colonization in a murine model of gonorrhea, suggesting that lactate utilization contributes to virulence. Gc encodes four lactate dehydrogenases (LDHs) with distinct regulation and mechanisms, including two L-LDHs, LldD and LutACB. Although either enzyme alone supports L-lactate utilization, we found that both are required for full fitness during co-colonization with PMNs, indicating some non-redundant roles. Furthermore, LldD, but not LutACB, enhances oxidative stress resistance and is required for Gc colonization in a murine model of gonorrhea, whereas LutACB is dispensable. These findings identify LldD as a key factor promoting oxidative stress resistance, survival during PMN challenge, and host colonization.

microbiology↗

bioRxiv2025

A Microphysiologic Model of the Cervical Epithelium Recapitulates Microbial, Immunologic, and Pathogenic Properties of Sexually Transmitted Infections

Sexually transmitted infections (STIs) of the cervicovaginal mucosa are among the most common global infections. Clinical studies have revealed that susceptibility to STIs and the subsequent host responses they elicit are frequently associated with vaginal microbiota compositions that facilitate infection. Current monolayer cell culture and animal models fail to reproduce the multilevel complexity required to investigate these relationships simultaneously and/or with sufficient physiological relevance. To address this limitation, we have developed a microphysiologic system (MPS) that models human cervical tissue, its microbiota, and is susceptible to infection by two prominent genital pathogens, Chlamydia trachomatis and Neisseria gonorrhoeae. Significantly, this MPS platform recapitulates essential dynamic, polymicrobial, immune, and pathogenic features of chlamydial and gonococcal infections as they occur in humans. The lowcost MPS device requires no specialized equipment or specific expertise and was experimentally validated for both chlamydial and gonococcal infections across multiple nonengineering, remotely located laboratories, demonstrating its transferability and reproducibility. The MPS platform described herein provides a novel tool for expanded research into genital infections in a reconstituted system that closely mimics the cervical epithelium, a significant advance over existing models.

bioengineering↗

bioRxiv2025

Terminal complement complexes with or without C9 potentiate antimicrobial activity against Neisseria gonorrhoeae

The complement cascade is a front-line defense against pathogens. Complement activation generates the membrane attack complex (MAC), a 10-11 nm diameter pore formed by complement proteins C5b through C8 and polymerized C9. The MAC embeds within the outer membrane of Gram-negative bacteria and displays bactericidal activity. In the absence of C9, C5b-C8 complexes can form 2-4 nm pores on membranes, but their relevance to microbial control is poorly understood. Deficiencies in terminal complement components uniquely predispose individuals to infections by pathogenic Neisseria, including N. gonorrhoeae (Gc). Increasing antibiotic resistance in Gc makes new therapeutic strategies a priority. Here, we demonstrate that MAC formed by complement activity in human serum disrupts the Gc outer and inner membranes, potentiating the activity of antimicrobials against Gc and re-sensitizing multidrug resistant Gc to antibiotics. C9-depleted serum also disrupts Gc membranes and exerts antigonococcal activity, effects that are not reported in other Gram-negative bacteria. C5b-C8 complex formation potentiates Gc sensitivity to azithromycin but not lysozyme. These findings expand our mechanistic understanding of complement lytic activity, suggest a size limitation for terminal complement-mediated enhancement of antimicrobials against Gc, and suggest complement manipulation can be used to combat drug-resistant gonorrhea. ImportanceThe complement cascade is a front-line arm of the innate immune system against pathogens. Complement activation results in membrane attack complex (MAC) pores forming on the outer membrane of Gram-negative bacteria, resulting in bacterial death. Individuals who cannot generate MAC are specifically susceptible to infection by pathogenic Neisseria species including N. gonorrhoeae (Gc). High rates of gonorrhea and its complications like infertility, and high-frequency resistance to multiple antibiotics, make it important to identify new approaches to combat Gc. Beyond direct anti-Gc activity, we found the MAC increases the ability of antibiotics and antimicrobial proteins to kill Gc and re-sensitizes multidrug-resistant bacteria to antibiotics. The most terminal component, C9, is needed to potentiate the anti-Gc activity of lysozyme, but azithromycin activity is potentiated regardless of C9. These findings highlight the unique effects of MAC on Gc and suggest novel translational avenues to combat drug-resistant gonorrhea.

microbiology↗

bioRxiv2024

High-dimensional spectral flow cytometry of activation and phagocytosis by peripheral human polymorphonuclear leukocytes

Polymorphonuclear lymphocytes (PMNs) are terminally differentiated phagocytes with pivotal roles in infection, inflammation, tissue injury, and resolution. PMNs can display a breadth of responses to diverse endogenous and exogenous stimuli, making understanding of these innate immune responders vital yet challenging to achieve. Here, we report a 22-color spectral flow cytometry panel to profile primary human PMNs on population and single cell levels for surface marker expression of activation, degranulation, phagocytosis, migration, chemotaxis, and interaction with fluorescently labeled cargo. We demonstrate the surface protein response of PMNs to phorbol ester stimulation compared to untreated controls in an adherent PMN model with additional analysis of intra- and inter-subject variability. PMNs challenged with the Gram-negative bacterial pathogen Neisseria gonorrhoeae revealed infectious dose-dependent changes in surface marker expression in bulk, population-level analysis. Imaging flow cytometry complemented spectral cytometry, demonstrating that fluorescence signal from labeled bacteria corresponded with bacterial burden on a per-cell basis. Spectral flow cytometry subsequently identified surface markers which varied with direct PMN-bacterium association as well as those which varied in the presence of bacteria but without phagocytosis. This spectral panel protocol highlights best practices for efficient customization and is compatible with downstream approaches such as spectral cell sorting and single-cell RNA-sequencing for applicability to diverse research questions in the field of PMN biology. Summary SentenceHere we report a 22-color spectral flow cytometry panel to profile primary human PMNs for markers of activation, degranulation, phagocytosis, migration, and chemotaxis using phorbol ester stimulation and bacterial challenge as proofs-of-concept.

immunology↗

bioRxiv2024

Protection against N. gonorrhoeae induced by OMV-based Meningococcal Vaccines are associated with cross-species directed humoral and cellular immune responses

Limited protective immunologic responses to natural N. gonorrhoeae infection and a lack of knowledge about mechanisms of protection have hampered development of an effective vaccine. Recent studies in humans and mice have found meningococcal outer membrane vesicle-containing vaccines (OMV) induce cross species immune responses against gonococci and are associated with protection. The exact mechanisms or how humoral and cellular immunity are related to protection, remain unclear. To study this, we immunized mice with two meningococcal OMV-containing vaccines known to accelerate clearance of N. gonorrhoeae, 4CMenB and OMV from an engineered N. meningitidis strain lacking major surface antigens PorA, PorB, and Rmp (MC58 {Delta}ABR). We assessed serologic and cellular immune signatures associated with these immunizations and assessed bacterial clearance in the mice using a vaginal/cervical gonococcal infection model. Mice immunized with 4CMenB or MC58 {Delta}ABR demonstrated shortened courses of recovery of vaginal N. gonorrhoeae compared to control mice immunized with alum alone. Vaccination with 4CMenB or MC58{Delta}ABR OMV elicited serum and vaginal cross-reactive anti-Ng-OMV antibody responses that were augmented after vaginal challenge with N. gonorrhoeae. Further, splenocytes in 4CMenB and MC58 {Delta}ABR immunized mice exhibited elevated cytokine production after restimulation with heterologous N. gonorrhoeae OMV when compared to splenocytes from Alum immunized mice. We further tested for correlations between bacterial burden and the measured anti-gonococcal immune responses within each vaccination group and found different immunologic parameters associated with reduced bacterial burden for each vaccine. Our findings suggest the cross-protection against gonococcal infection induced by different meningococcal OMV vaccines is likely multifactorial and mediated by different humoral and cellular immune responses induced by these two vaccines.

microbiology↗

bioRxiv2024

Neisseria gonorrhoeae scavenges host sialic acid for Siglec-mediated, complement-independent suppression of neutrophil activation

Gonorrhea, caused by the bacterium Neisseria gonorrhoeae (Gc), is characterized by neutrophil influx to infection sites. Gc has developed mechanisms to resist killing by neutrophils that include modifications to its surface lipooligosaccharide (LOS). One such LOS modification is sialylation: Gc sialylates its terminal LOS sugars with cytidine-5-monophosphate-N-acetylneuraminic acid (CMP-NANA) scavenged from the host using LOS sialyltransferase (Lst), since Gc cannot make its own sialic acid. Sialylation enables sensitive strains of Gc to resist complement-mediated killing in a serum-dependent manner. However, little is known about the contribution of sialylation to complement-independent, direct Gc-neutrophil interactions. In the absence of complement, we found sialylated Gc expressing opacity-associated (Opa) proteins decreased the oxidative burst and granule exocytosis from primary human neutrophils. In addition, sialylated Opa+ Gc survived better than vehicle treated or {Delta}lst Gc when challenged with neutrophils. However, Gc sialylation did not significantly affect Opa-dependent association with or internalization of Gc by neutrophils. Previous studies have implicated sialic acid-binding immunoglobulin-type lectins (Siglecs) in modulating neutrophil interactions with sialylated Gc. Blocking neutrophil Siglecs with antibodies that bind to their extracellular domains eliminated the ability of sialylated Opa+ Gc to suppress oxidative burst and resist neutrophil killing. These findings highlight a new role for sialylation in Gc evasion of human innate immunity, with implications for the development of vaccines and therapeutics for gonorrhea. IMPORTANCENeisseria gonorrhoeae, the bacterium that causes gonorrhea, is an urgent global health concern due to increasing infection rates, widespread antibiotic resistance, and its ability to thwart protective immune responses. The mechanisms by which Gc subvert protective immune responses remain poorly characterized. One way N. gonorrhoeae evades human immunity is by adding sialic acid that is scavenged from the host onto its lipooligosaccharide, using the sialyltransferase Lst. Here, we found that sialylation enhances N. gonorrhoeae survival from neutrophil assault and inhibits neutrophil activation, independently of the complement system. Our results implicate bacterial binding of sialic acid-binding lectins (Siglecs) on the neutrophil surface, which dampen neutrophil antimicrobial responses. This work identifies a new role for sialylation in protecting N. gonorrhoeae from cellular innate immunity, which can be targeted to enhance the human immune response in gonorrhea.

microbiology↗

bioRxiv2023

Evaluating vaccine-elicited antibody activities against Neisseria gonorrhoeae: cross-protective responses elicited by the 4CMenB meningococcal vaccine

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.

microbiology↗

bioRxiv2022

Transcriptome guided metabolic network analysis reveals rearrangements of carbon flux distribution in Neisseria gonorrhoeae during neutrophil co-culture

The ability of bacterial pathogens to metabolically adapt to the environmental conditions of their hosts is critical to both colonization and invasive disease. Infection with Neisseria gonorrhoeae (the gonococcus, Gc) is characterized by the influx of neutrophils (PMNs), which fail to clear the bacteria and make antimicrobial products that can exacerbate tissue damage. The inability of the human host to clear Gc infection is particularly concerning in light of the emergence of strains that are resistant to all clinically recommended antibiotics. Bacterial metabolism represents a promising target for the development of new therapeutics against Gc. Here, we generated a curated genome-scale metabolic network reconstruction (GENRE) of Gc strain FA1090. This GENRE links genetic information to metabolic phenotypes and predicts Gc biomass synthesis and energy consumption. We validated this model with published data and in new results reported here. Contextualization of this model using the transcriptional profile of Gc exposed to PMNs revealed substantial rearrangements of Gc central metabolism and induction of Gc nutrient acquisition strategies for alternate carbon source use. These features enhanced the growth of Gc in the presence of neutrophils. From these results we conclude that the metabolic interplay between Gc and PMNs helps define infection outcomes. The use of transcriptional profiling and metabolic modeling to reveal new mechanisms by which Gc persists in the presence of PMNs uncovers unique aspects of metabolism in this fastidious bacterium, which could be targeted to block infection and thereby reduce the burden of gonorrhea in the human population. ImportanceThe World Health Organization (WHO) designated Neisseria gonorrhoeae (Gc) as a high priority pathogen for research and development of new antimicrobials. Bacterial metabolism is a promising target for new antimicrobials, as metabolic enzymes are widely conserved among bacterial strains and are critical for nutrient acquisition and survival within the human host. Here we used genome-scale metabolic modeling to characterize the core metabolic pathways of this fastidious bacterium, and to uncover the pathways used by Gc during culture with primary human immune cells. These analyses revealed that Gc relies on different metabolic pathways during co-culture with human neutrophils than in rich media. Conditionally essential genes emerging from these analyses were validated experimentally. These results show that metabolic adaptation in the context of innate immunity is important to Gc pathogenesis. Identifying the metabolic pathways used by Gc during infection can highlight new therapeutic targets for drug-resistant gonorrhea.

microbiology↗

bioRxiv2022

Neisseria gonorrhoeae co-opts C4b-binding protein to enhance complement-independent survival from neutrophils

Neisseria gonorrhoeae (Gc) is a human-specific pathogen that causes the sexually transmitted infection gonorrhea. Gc survives in neutrophil-rich gonorrheal secretions, and recovered bacteria predominantly express phase-variable, surface-expressed opacity-associated (Opa) proteins (Opa+). However, expression of Opa proteins like OpaD decreases Gc survival when exposed to human neutrophils ex vivo. Here, we made the unexpected observation that incubation with normal human serum, which is found in inflamed mucosal secretions, enhances survival of Opa+ Gc from primary human neutrophils. We directly linked this phenomenon to a novel complement-independent function for C4b-binding protein (C4BP). When bound to the bacteria, C4BP was necessary and sufficient to suppress Gc-induced neutrophil reactive oxygen species production and prevent neutrophil phagocytosis of Opa+ Gc. This research identifies for the first time a complement-independent role for C4BP in enhancing the survival of a pathogenic bacterium from phagocytes, thereby revealing how Gc exploits inflammatory conditions to persist at human mucosal surfaces. Author SummaryGonorrhea is considered an urgent threat to public health with an estimated 98 million cases occurring annually worldwide, growing antimicrobial resistance, and the absence of a gonococcal vaccine. Currently, we do not understand how N. gonorrhoeae expressing opacity (Opa) proteins survive neutrophil defenses and are recovered viable from infected patients. Here, we investigated how soluble elements of gonorrhea infection, present in human serum, contribute to N. gonorrhoeae survival from neutrophils. We found that the serum component C4b-binding protein (C4BP) protects N. gonorrhoeae from neutrophil killing and suppresses neutrophil activation. C4BP limited neutrophil phagocytosis of N. gonorrhoeae that expressed Opa proteins that bound to neutrophil receptors of the CEACAM family. This work provides novel insight into the interplay between the noncellular and cellular aspects of the innate immune response to N. gonorrhoeae.

microbiology↗

bioRxiv2022

Phagocytosis via complement receptor 3 enables microbes to evade killing by neutrophils

Complement receptor 3 (CR3; CD11b/CD18; m{beta}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 ({Delta}opa) 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 {Delta}opa 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 {Delta}opa Ngo. Neutrophils exposed to {Delta}opa 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.

microbiology↗

bioRxiv2022

Dual species transcriptomics reveals core metabolic and immunologic processes in the interaction between primary human neutrophils and Neisseria gonorrhoeae strains

Neisseria gonorrhoeae (the gonococcus, Gc) is the causative agent of the sexually transmitted infection gonorrhea. Gc is a prominent threat to human health by causing severe and lifelong clinical sequelae, including infertility and chronic pelvic pain, which is amplified by the emergence of "superbug" strains that are resistant to all current antibiotics. Gc is highly adapted to colonize human mucosal surfaces, where it survives despite initiating a robust inflammatory response and influx of polymorphonuclear leukocytes (PMNs or neutrophils) that typically clear bacteria. Here, dual-species RNA-sequencing (RNA-seq) was used to define Gc and PMN transcriptional profiles alone and after infection. Three strains of Gc and three human donors transcriptional responses were assessed to characterize core host and bacterial responses. Comparative analysis of Gc transcripts revealed major overlap between the Gc response to PMNs, iron, and hydrogen peroxide; specifically, the TonB system and TonB dependent transporters (TDT) were upregulated in response to PMNs. We experimentally confirmed that induction of the iron-dependent TDT TbpB is responsive to the presence of PMNs and that tonB is required for Gc survival from PMNs. Pathway analysis of PMN transcripts induced by Gc infection revealed differential expression of genes driving pathways involved in cell adhesion and migration, inflammatory responses, and inflammation resolution. Production of pro-inflammatory cytokines, including IL1B and IL8, the adhesion factor ICAM1, and the anti-inflammatory prostaglandin PGE2 was confirmed to be induced in PMNs in response to Gc. Together, this study represents a comprehensive and experimentally validated dual-species transcriptomic analysis of three isolates of Gc and primary human PMNs that gives insight into how this bacterium survives innate immune onslaught to cause disease in humans.

genomics↗