Validation for the function of protein C in mouse models.

Objectives: Protein C (PC) is an anticoagulant that is encoded by the PROC gene. Validation for the function of PC was carried out in mouse models. Methods: In this study, autosomal recessive PC deficiency (PCD) was selected as the target, and the specific mutation site was chromosome 2 2q13-q14, PROC c.1198G>A (p.Gly400Ser) which targets G399S (GGT to AGC) in mouse models. To investigate the role of hereditary PC in mice models, we used CRISPR/Cas9 gene editing technology to create a mouse model with a genetic PCD mutation. Results: The two F0 generation positive mice produced using the CRISPR/Cas9 gene editing technique were chimeras, and the mice in F1 and F2 generations were heterozygous. There was no phenotype of spontaneous bleeding or thrombosis in the heterozygous mice, but some of them were blind. Blood routine results showed no significant difference between the heterozygous mice and wild-type mice (P > 0.05). Prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT) were prolonged in the heterozygous mice, while the level of fibrinogen content (FIB) decreased, suggesting secondary consumptive coagulation disease. The protein C activity of heterozygous mice was significantly lower than that of wild-type mice (P < 0.001), but there was no significant difference in protein C antigen levels (P > 0.05). H&E staining showed steatosis and hydrodegeneration in the liver of heterozygous mice. Necrosis and exfoliated epithelial cells could be observed in renal tubule lumen, forming cell or granular tubules. Hemosiderin deposition was found in the spleen along with splenic hemorrhage. Immunohistochemistry demonstrated significant fibrin deposition in the liver, spleen, and kidney of heterozygous mice. Conclusion: In this study, heterozygotes of the mouse model with a PC mutation were obtained. The function of PC was then validated in a mouse model through genotype, phenotype, and PC function analysis.

A common protein C inhibitor exosite partially controls the heparin induced activation and inhibition of serine proteases.

Protein C inhibitor (PCI) maintains hemostasis by inhibiting both procoagulant and anticoagulant serine proteases, and plays important roles in coagulation, fibrinolysis, reproduction, and anti-angiogenesis. The reactive site loop of PCI traps and irreversibly inhibits the proteases like APC (activating protein C), thrombin (FIIa) and factor Xa (FXa). Previous studies on antithrombin (ATIII) had identified Tyr253 and Glu255 as functional exosites that interact and aid in the inhibition of factor IXa and FXa. Presence of exosite in PCI is not known, however a sequence comparison with the PCI from different vertebrate species and ATIII identified Glu239 to be absolutely conserved. PCI residues analogous to ATIII exosite residues were mutated to R238A and E239A. Purified variant PCI in the presence of heparin (10 µg/ml) showed a 2-4 fold decrease in the rate of inhibition of the proteases. However, the stoichiometry of inhibition of FIIa, APC, and FXa by native PCI, R238A and E239A variants were found to be close to 1.0, which also indicated the formation of stable complexes based on SDS-PAGE and western blot analysis with thrombin and APC. Our findings revealed the possible presence of an exosite in PCI that influences the protease inhibition rates.

A potential mechanism for the cytoprotective effects of activated protein C-released endothelial extracellular vesicles.

ABSTRACT: Activated protein C (APC) was shown to release extracellular vesicles (EVs). APC bound to the EVs was thought to be responsible for cytoprotection. Our study demonstrates that the cytoprotective effects of APC-released EVs are independent of APC. APC-released EVs carry anti-inflammatory microRNAs in their cargo.

N-Terminal Fragment of Cardiac Myosin Binding Protein C Modulates Cooperative Mechanisms of Thin Filament Activation in Atria and Ventricles.

Cardiac myosin binding protein C (cMyBP-C) is one of the essential control components of the myosin cross-bridge cycle. The C-terminal part of cMyBP-C is located on the surface of the thick filament, and its N-terminal part interacts with actin, myosin, and tropomyosin, affecting both kinetics of the ATP hydrolysis cycle and lifetime of the cross-bridge, as well as calcium regulation of the actin-myosin interaction, thereby modulating contractile function of myocardium. The role of cMyBP-C in atrial contraction has not been practically studied. We examined effect of the N-terminal C0-C1-m-C2 (C0-C2) fragment of cMyBP-C on actin-myosin interaction using ventricular and atrial myosin in an in vitro motility assay. The C0-C2 fragment of cMyBP-C significantly reduced the maximum sliding velocity of thin filaments on both myosin isoforms and increased the calcium sensitivity of the actin-myosin interaction. The C0-C2 fragment had different effects on the kinetics of ATP and ADP exchange, increasing the affinity of ventricular myosin for ADP and decreasing the affinity of atrial myosin. The effect of the C0-C2 fragment on the activation of the thin filament depended on the myosin isoforms. Atrial myosin activates the thin filament less than ventricular myosin, and the C0-C2 fragment makes these differences in the myosin isoforms more pronounced.

Single-Cell RNA-seq reveals transcriptomic modulation of Alzheimer's disease by activated protein C.

Single-Cell RNA sequencing reveals changes in cell population in Alzheimer's disease (AD) model 5xFAD (5x Familial AD mutation) versus wild type (WT) mice. The returned sequencing data was processed through the 10x Genomics CellRanger platform to perform alignment and form corresponding matrix to perform bioinformatic analysis. Alterations in glial cells occurred in 5xFAD versus WT, especially increases in microglia proliferation were profound in 5xFAD. Differential expression testing of glial cells in 5xFAD versus WT revealed gene regulation. Globally, the critical genes implicated in AD progression are upregulated such as Apoe, Ctsb, Trem2, and Tyrobp. Using this differential expression data, GO term enrichment was completed to observe possible biological processes impacted by AD progression. Utilizing anti-inflammatory and cyto-protective recombinant Activated Protein C (APC), we uncover inflammatory processes to be downregulated by APC treatment in addition to recuperation of nervous system processes. Moreover, animal studies demonstrated that administration of recombinant APC significantly attenuated Aß burden and improved cognitive function of 5xFAD mice. The downregulation of highly expressed AD biomarkers in 5xFAD could provide insight into the mechanisms by which APC administration benefits AD.

Thrombomodulin: a multifunctional receptor modulating the endothelial quiescence.

Thrombomodulin (TM) is a type 1 receptor best known for its function as an anticoagulant cofactor for thrombin activation of protein C on the surface of vascular endothelial cells. In addition to its anticoagulant cofactor function, TM also regulates fibrinolysis, complement, and inflammatory pathways. TM is a multidomain receptor protein with a lectin-like domain at its N-terminus that has been shown to exhibit direct anti-inflammatory functions. This domain is followed by 6 epidermal growth factor-like domains that support the interaction of TM with thrombin. The interaction inhibits the procoagulant function of thrombin and enables the protease to regulate the anticoagulant and fibrinolytic pathways by activating protein C and thrombin-activatable fibrinolysis inhibitor. TM has a Thr/Ser-rich region immediately above the membrane surface that harbors chondroitin sulfate glycosaminoglycans, and this region is followed by a single-spanning transmembrane and a C-terminal cytoplasmic domain. The structure and physiological function of the extracellular domains of TM have been extensively studied, and numerous excellent review articles have been published. However, the physiological function of the cytoplasmic domain of TM has remained poorly understood. Recent data from our laboratory suggest that intracellular signaling by the cytoplasmic domain of TM plays key roles in maintaining quiescence by modulating phosphatase and tensin homolog signaling in endothelial cells. This article briefly reviews the structure and function of extracellular domains of TM and focuses on the mechanism and possible physiological importance of the cytoplasmic domain of TM in modulating phosphatase and tensin homolog signaling in endothelial cells.

Endothelial Protein C Receptor and 3K3A-Activated Protein C Protect Mice from Allergic Contact Dermatitis in a Contact Hypersensitivity Model.

Endothelial protein C receptor (EPCR) is a receptor for the natural anti-coagulant activated protein C (aPC). It mediates the anti-inflammatory and barrier-protective functions of aPC through the cleavage of protease-activated receptor (PAR)1/2. Allergic contact dermatitis is a common skin disease characterized by inflammation and defective skin barrier. This study investigated the effect of EPCR and 3K3A-aPC on allergic contact dermatitis using a contact hypersensitivity (CHS) model. CHS was induced using 1-Fluoro-2,4-dinitrobenzene in EPCR-deficient (KO) and matched wild-type mice and mice treated with 3K3A-aPC, a mutant form of aPC with diminished anti-coagulant activity. Changes in clinical and histological features, cytokines, and immune cells were examined. EPCRKO mice displayed more severe CHS, with increased immune cell infiltration in the skin and higher levels of inflammatory cytokines and IgE than wild-type mice. EPCR, aPC, and PAR1/2 were expressed by the skin epidermis, with EPCR presenting almost exclusively in the basal layer. EPCRKO increased the epidermal expression of aPC and PAR1, whereas in CHS, their expression was reduced compared to wild-type mice. 3K3A-aPC reduced CHS severity in wild-type and EPCRKO mice by suppressing immune cell infiltration/activation and inflammatory cytokines. In summary, EPCRKO exacerbated CHS, whereas 3K3A-aPC could reduce the severity of CHS in both EPCRKO and wild-type mice.

Anticoagulant effects of protein C, protein S, and antithrombin levels on the protein C pathway in young children.

The protein C (PC) pathway involves physiological anticoagulant factors (PC, protein S [PS], and factor V) and performs major anticoagulant functions in adults. Variations in overall PC pathway function due to dynamic changes in PC and PS in early childhood are poorly understood. We aimed to evaluate the contributions of PC pathway function during early childhood by measuring changes in plasma thrombin generation (TG) after administration of the PC activator protac. We evaluated correlations between anticoagulant factors and percentage of protac-induced coagulation inhibition (PiCi%). Before protac addition, TG in newborns (n = 35), infants (n = 42), young children (n = 35), and adults (n = 20) were 525 ± 74, 720 ± 96, 785 ± 53, and 802 ± 64 mOD/min, and PiCi% were 42.1 ± 9.9, 69.8 ± 11.0, 82.9 ± 4.4, and 86.9 ± 3.4%, respectively. The distribution of PiCi% on the two axes of TG (with or without protac) changed continuously with age and differed from that of warfarin-treated plasma and adult PC- or PS-deficient plasma. PiCi% increased dynamically during infancy and correlated with PS levels in newborns and PC levels in young children. Addition of PC or fresh frozen plasma equivalent to approximately 25% PC to PC-deficient plasma improved PiCi%. This automatic measurement requires only a small sample volume and is useful for analysis of developmental hemostasis.

Structural vulnerability in EPCR suggests functional modulation.

The endothelial protein C receptor (EPCR) is a fundamental component of the vascular system in mammals due to its contribution in maintaining blood in a non-prothrombotic state, which is crucial for overall life development. It accomplishes this by enhancing the conversion of protein C (PC) into the anticoagulant activated protein C (APC), with this property being dependent on a known EPCR conformation that enables direct interaction with PC/APC. In this study, we report a previously unidentified conformation of EPCR whereby Tyr154, critical for PC/APC binding, shows a striking non-canonical configuration. This unconventional form is incompatible with PC/APC binding, and reveals, for the first time, a region of structural vulnerability and potential modulation in EPCR. The identification of this malleability enhances our understanding of this receptor, prompting inquiries into the interplay between its plasticity and function, as well as its significance within the broader framework of EPCR's biology, which extends to immune conditions.

Hemostatic balance between pro- and anticoagulant is maintained during glucocorticoid treatment.

BACKGROUND: Glucocorticoids are associated with an increased risk of venous thrombosis. Glucocorticoid treatment increases coagulation factor and anticoagulant levels; however, its effect on hemostatic function remains unclear. This study aimed to investigate the changes in comprehensive coagulation profiles after glucocorticoid treatment in noninflammatory diseases to elucidate the direct contribution of glucocorticoids to hemostatic function. PROCEDURE: Patients diagnosed with primary immune thrombocytopenia requiring glucocorticoid treatment were prospectively enrolled in this study. Changes in coagulation factors and anticoagulants during glucocorticoid treatment and changes in thrombin generation potential were determined in the absence and presence of soluble thrombomodulin (sTM). RESULTS: Seven treatment cases (four for steroid pulse therapy and three for oral glucocorticoid therapy) in six patients with immune thrombocytopenia were examined. After glucocorticoid treatment, activated partial thromboplastin time significantly shortened, and activities of factor VIII, IX, XI, and XII significantly increased, except for von Willebrand factor antigen. Moreover, antithrombin and protein C (PC) activities significantly increased after glucocorticoid treatment. Two major parameters of thrombin generation potential, endogenous thrombin potential (ETP) and peak thrombin (Peak), significantly increased in the absence of sTM after glucocorticoid treatment. However, no significant increases in either parameter were observed in the presence of sTM. ETP-TM and Peak-TM ratios, which represent resistance to the anticoagulant effect of the PC pathway, significantly decreased after glucocorticoid treatment, suggesting that anticoagulant function via the PC pathway is elevated after glucocorticoid treatment. CONCLUSIONS: As glucocorticoids increase intrinsic coagulation factor and anticoagulant levels, hemostatic balance between pro- and anticoagulant functions is maintained.

Analysis of PROC mutations and clinical features in 22 unrelated families with inherited protein C deficiency.

Currently, limited information is available in the literature regarding the relationships between PROC mutations and clinical features in Chinese individuals. We aimed to characterize severe congenital Protein C deficiency in 22 unrelated Chinese families in a tertiary hospital by analyzing its clinical manifestation, associated risk factors, and gene mutations. We measured protein C activity and antigen levels for all participants, screened them for mutations in the PROC gene, and analyzed the clinical features of each family to identify commonalities and differences. The analysis revealed a total of 75 individuals with PCD and 16 different PROC mutations, including 12 missense mutations and 4 deletion mutations. Among them, 11 who were compound heterozygotes or homozygotes for mutations tended to develop symptoms at a younger age without any clear triggers. In contrast, the remaining 64 individuals who were heterozygotes for mutations often had clear triggers for their symptoms and experienced a milder course of the disease. It is worth noting that the mutation c.565C > T occurred most frequently, being identified in 8 out of 22 families (36%). Our team also reported five novel mutations, including c.742-744delAAG, c.383G > A, c.997G > A, c.1318C > T, and c.833T > C mutations. The identification of five novel mutations adds to the richness of the Human Genome Database. Asymptomatic heterozygotes are not uncommon, and they are prone to develop symptoms with obvious triggers. The evidence presented strongly suggest that asymptomatic individuals with family history of protein C deficiency can benefit from mutational analysis of PROC gene.

Discovery of plant-based phytochemical as effective antivirals that target the non-structural protein C of the Nipah virus through computational methods.

Nipah Virus (NiV) belongs to the Paramyxoviridae family and was first identified during an outbreak in Malaysia. Some initial symptoms include mild fever, headache and sore throat, which could escalate to respiratory illness and brain inflammation. The mortality rate of NiV infection can range from 40% to 75%, which is quite high. This is mainly due to the lack of efficient drugs and vaccines. In most instances, NiV is transmitted from animals to humans. Non-Structural Proteins (C, V and W) of the Nipah virus impede the host immune response by obstructive the JAK/STAT pathway. However, Non-Structural Proteins - C (NSP-C) plays a vital role in NiV pathogenesis, which includes IFN antagonist activity and viral RNA production. In the present study, the full-length structure of NiV-NSP-C was predicted using computational modelling, and the stability of the structure was analysed using 200 ns molecular dynamic (MD) simulation. Further, the structure-based virtual screening identified five potent phytochemicals (PubChem CID: 9896047, 5885, 117678, 14887603 and 5461026) with better binding affinity against NiV-NSP-C. DFT studies clearly showed that the phytochemicals had higher chemical reactivity, and the complex MD simulation depicted that the identified inhibitors exhibited stable binding with NiV-NSP-C. Furthermore, experimental validation of these identified phytochemicals would likely control the infection of NiV.Communicated by Ramaswamy H. Sarma.

Mitigation of trauma-induced endotheliopathy by activated protein C: A potential therapeutic for postinjury thromboinflammation.

BACKGROUND: Activated Protein C (aPC) plays dual roles after injury, driving both trauma-induced coagulopathy (TIC) by cleaving, and thus inactivating, factors Va and VIIIa and depressing fibrinolysis while also mediating an inflammomodulatory milieu via protease activated receptor-1 (PAR-1) cytoprotective signaling. Because of this dual role, it represents and ideal target for study and therapeutics after trauma. A known aPC variant, 3K3A-aPC, has been engineered to preserve cytoprotective activity while retaining minimal anticoagulant activity rendering it potentially ideal as a cytoprotective therapeutic after trauma. We hypothesized that 3K3A-aPC would mitigate the endotheliopathy of trauma by protecting against endothelial permeability. METHODS: We used electric cell-substrate impedance sensing to measure permeability changes in real time in primary endothelial cells. These were cultured, grown to confluence, and treated with a 2 µg/mL solution of 3K3A-aPC at 180 minutes, 120 minutes, 60 minutes, 30 minutes prior to stimulation with ex vivo plasma taken from severely injured trauma patients (Injury Severity Score > 15 and BD < -6) (trauma plasma [TP]). Cells treated with thrombin and untreated cells were included in this study as control groups. Permeability changes were recorded in real time via electric cell-substrate impedance sensing for 30 minutes after treatment with TP. We quantified permeability changes in the control and treatment groups as area under the curve (AUC). Rac1/RhoA activity was also compared between these groups. Statistical significance was determined by one-way ANOVA followed by a post hoc analysis using Tukey's multiple comparison's test. RESULTS: Treatment with aPC mitigated endothelial permeability induced by ex vivo trauma plasma at all pre-treatment time points. The AUC of the 30-minute 3K3A-aPC pretreatment group was higher than TP alone (mean diff. 22.12 95% CI [13.75, 30.49], p < 0.0001) (Figure). Moreover, the AUC of the 60-minute, 120-minute, and 180-minute pretreatment groups was also higher than TP alone (mean diff., 16.30; 95% confidence interval [CI], 7.93-24.67; 19.43; 95% CI, 11.06-27.80, and 18.65; 95% CI, 10.28-27.02;, all p < 0.0001, respectively). Rac1/RhoA activity was higher in the aPC pretreatment group when compared with all other groups ( p < 0.01). CONCLUSION: Pretreatment with 3K3A-aPC, which retains its cytoprotective function but has only ~5% of its anticoagulant function, abrogates the effects of trauma-induced endotheliopathy. This represents a potential therapeutic treatment for dysregulated thromboinflammation for injured patients by minimizing aPC's role in trauma-induced coagulopathy while concurrently amplifying its essential cytoprotective function. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level III.

Glycolytic reprogramming fuels myeloid cell-driven hypercoagulability.

BACKGROUND: Myeloid cell metabolic reprogramming is a hallmark of inflammatory disease; however, its role in inflammation-induced hypercoagulability is poorly understood. OBJECTIVES: We aimed to evaluate the role of inflammation-associated metabolic reprogramming in regulating blood coagulation. METHODS: We used novel myeloid cell-based global hemostasis assays and murine models of immunometabolic disease. RESULTS: Glycolysis was essential for enhanced activated myeloid cell tissue factor expression and decryption, driving increased cell-dependent thrombin generation in response to inflammatory challenge. Similarly, inhibition of glycolysis enhanced activated macrophage fibrinolytic activity through reduced plasminogen activator inhibitor 1 activity. Macrophage polarization or activation markedly increased endothelial protein C receptor (EPCR) expression on monocytes and macrophages, leading to increased myeloid cell-dependent protein C activation. Importantly, inflammation-dependent EPCR expression on tissue-resident macrophages was also observed in vivo. Adipose tissue macrophages from obese mice fed a high-fat diet exhibited significantly enhanced EPCR expression and activated protein C generation compared with macrophages isolated from the adipose tissue of healthy mice. Similarly, the induction of colitis in mice prompted infiltration of EPCR+ innate myeloid cells within inflamed colonic tissue that were absent from the intestinal tissue of healthy mice. CONCLUSION: Collectively, this study identifies immunometabolic regulation of myeloid cell hypercoagulability, opening new therapeutic possibilities for targeted mitigation of thromboinflammatory disease.

Thrombin has dual trypsin-like and chymotrypsin-like specificity.

BACKGROUND: The residue at the site of activation of protein C is Arg in all species except the ray-finned fish, where it is Trp. This feature raises the question of whether thrombin is the physiological activator of protein C across vertebrates. OBJECTIVES: To establish if thrombin can cleave at Trp residues. METHODS: The activity of wild-type thrombin and mutant D189S was tested with a library of chromogenic substrates and toward wild-type protein C and mutants carrying substitutions at the site of cleavage. RESULTS: Thrombin has trypsin-like and chymotrypsin-like specificity and cleaves substrates at Arg or Trp residues. Cleavage at Arg is preferred, but cleavage at Trp is significant and comparable with that of chymotrypsin. The D189S mutant of thrombin has broad specificity and cleaves at basic and aromatic residues without significant preference. Thrombin also cleaves natural substrates at Arg or Trp residues, showing activity toward protein C across vertebrates, including the ray-finned fish. The rate of activation of protein C in the ray-finned fish is affected by the sequence preceding Trp at the scissile bond. CONCLUSION: The results provide a possible solution for the paradoxical presence of a Trp residue at the site of cleavage of protein C in ray-finned fish and support thrombin as the physiological activator of protein C in all vertebrates. The dual trypsin-like and chymotrypsin-like specificity of thrombin suggests that the spectrum of physiological substrates of this enzyme is broader currently assumed.

A recombinant signalling-selective activated protein C that lacks anticoagulant activity is efficacious and safe in cutaneous wound preclinical models.

Various preclinical and clinical studies have demonstrated the robust wound healing capacity of the natural anticoagulant activated protein C (APC). A bioengineered APC variant designated 3K3A-APC retains APC's cytoprotective cell signalling actions with <10% anticoagulant activity. This study was aimed to provide preclinical evidence that 3K3A-APC is efficacious and safe as a wound healing agent. 3K3A-APC, like wild-type APC, demonstrated positive effects on proliferation of human skin cells (keratinocytes, endothelial cells and fibroblasts). Similarly it also increased matrix metollaproteinase-2 activation in keratinocytes and fibroblasts. Topical 3K3A-APC treatment at 10 or 30 µg both accelerated mouse wound healing when culled on Day 11. And at 10 µg, it was superior to APC and had half the dermal wound gape compared to control. Further testing was conducted in excisional porcine wounds due to their congruence to human skin. Here, 3K3A-APC advanced macroscopic healing in a dose-dependent manner (100, 250 and 500 µg) when culled on Day 21. This was histologically corroborated by greater collagen maturity, suggesting more advanced remodelling. A non-interference arm of this study found no evidence that topical 3K3A-APC caused either any significant systemic side-effects or any significant leakage into the circulation. However the female pigs exhibited transient and mild local reactions after treatments in week three, which did not impact healing. Overall these preclinical studies support the hypothesis that 3K3A-APC merits future human wound studies.

C-terminal modification and functionalization of proteins via a self-cleavage tag triggered by a small molecule.

The precise modification or functionalization of the protein C-terminus is essential but full of challenges. Herein, a chemical approach to modify the C-terminus is developed by fusing a cysteine protease domain on the C-terminus of the protein of interest, which could achieve the non-enzymatic C-terminal functionalization by InsP6-triggered cysteine protease domain self-cleavage. This method demonstrates a highly efficient way to achieve protein C-terminal functionalization and is compatible with a wide range of amine-containing molecules and proteins. Additionally, a reversible C-terminal de-functionalization is found by incubating the C-terminal modified proteins with cysteine protease domain and InsP6, providing a tool for protein functionalization and de-functionalization. Last, various applications of protein C-terminal functionalization are provided in this work, as demonstrated by the site-specific assembly of nanobody drug conjugates, the construction of a bifunctional antibody, the C-terminal fluorescent labeling, and the C-terminal transpeptidation and glycosylation.

Negligible role for pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC) in the nasopharyngeal colonization of mice with a serotype 6B pneumococcal strain.

Streptococcus pneumoniae colonizes the human nasopharynx asymptomatically, but it can also cause several diseases, including otitis media, pneumonia, bacteremia, and meningitis. The colonization of the nasopharynx by the bacteria is an essential step for the pneumococcus to invade other sites and cause diseases. Pneumococcal surface protein A (PspA) and Pneumococcal surface Protein C (PspC) are important virulence factors and have been described to play roles in adhesion and immune evasion. In this study, we immunized mice subcutaneously with the recombinant α-helical region of PspA and/or PspC combined with different adjuvants to assess protection against colonization with the serotype 6B strain BHN418. Though high serum levels of specific IgG were detected, none of the formulations led to reduction in the colonization of the nasopharynx. The negative result may be due to the poor induction of IgG2c, which has been previously correlated with protection against pneumococcal colonization in mice. Furthermore, BHN418 pspA and pspC single and double knockouts were evaluated in colonization experiments and no differences in bacterial load were observed. In competition assays with the wild-type strain, borderline to no reduction was observed in the loads of the knockouts. Our results contrast with data from the literature using other pneumococcal strains, showing that the role of PspA and PspC in colonization can vary depending on the background of the knockout strain studied. BHN418 has been selected for its capacity to colonize humans in experimental challenge studies and may have redundant factors that compensate for the lack of PspA and PspC during nasopharyngeal colonization of mice.