ABSTRACT Ehrlichia chaffeensis is a tick-borne obligate intracellular bacterium that causes human monocytic ehrlichiosis. Significant advances have been made in understanding immune evasion strategies in Ehrlichia ; however, the role of the complement system has been largely overlooked. This study provides evidence for a novel interaction between E. chaffeensis and the complement inhibitor C4b-binding protein (C4BP). We confirmed that E. chaffeensis recruits and binds C4BP at its surface, demonstrating binding of C4BP in human serum. This interaction is concentration-dependent, and C4BP retains its regulatory cofactor activity when bound to the bacteria. Furthermore, we identified the ehrlichial retropepsin-type protease—named APEhc—as a new C4BP ligand. We show that the soluble domain of APEhc targets multiple serum components, directly binding factor H, forming complexes with C4BP, and cleaving two major complement components, C3b and C4b. Quantitative binding analysis reveals a strong interaction between APEhc and C4BP, with apparent dissociation constants in the nanomolar range. Analysis of C4BP CCP deletion mutants supports a contribution of CCP3, CCP6, and CCP7 to APEhc-C4BP binding. Additionally, we demonstrate that C4BP retains its factor I cofactor activity in the presence of APEhc, and that APEhc independently cleaves C4b. Consistent with the targeting and modulation of serum components, our results show that APEhc significantly inhibits activation of the classical, lectin, and alternative pathways. Our findings reveal the unprecedented interaction between E. chaffeensis , its retropepsin protease, and complement inhibitors, raising intriguing questions regarding complement targeting by E. chaffeensis and its role in immune evasion. IMPORTANCE Human monocytic ehrlichiosis is a tick-borne disease caused by Ehrlichia chaffeensis , which can be severe or fatal if untreated. Understanding how E. chaffeensis evades the immune system is crucial for identifying new virulence factors and developing new treatments. The complement system, an essential part of the body’s first line of defense, is often targeted by pathogens to avoid immune attacks. We show that E. chaffeensis binds to C4b-binding protein (C4BP), a major inhibitor of the classical pathway (CP) and lectin pathway (LP), and identify the ehrlichial retropepsin-type protease, APEhc, as a novel C4BP ligand. Additionally, we demonstrate that APEhc targets other serum components, inhibiting complement CP, LP, and AP activation, suggesting an unprecedented dual-inhibitory activity for APEhc through complex formation with C4BP (and factor H) and cleavage of C3b and C4b. Our findings reveal a multifunctional role for APEhc in immune evasion, raising intriguing questions about the mechanisms used by intracellular pathogens like E. chaffeensis to bypass host defenses.
ABSTRACT Sterylglucosidases are fungus-specific enzymes that regulate ergosterol 3-β-D-glucoside (Erg-SG) homeostasis and represent promising targets for antifungal intervention. Previous studies demonstrated that sterylglucosidases from Cryptococcus neoformans and Aspergillus fumigatus contribute to fungal virulence, and that deletion of these enzymes elicits protective immunity. However, whether sterylglucosidase-mediated pathways similarly influence Candida albicans pathogenicity remains unknown. Here, we identified C. albicans Sgl1 as a bona fide sterylglucosidase that selectively hydrolyzes Erg-SG but not fungal glucosylceramide. Deletion of SGL1 resulted in marked Erg-SG accumulation without altering glucosylceramide levels, confirming its conserved role in sterylglucoside homeostasis. Although the Δ sgl 1 mutant displayed largely preserved growth, morphogenesis, and stress adaptation, it exhibited impaired biofilm formation. Biochemical characterization, inhibitor profiling, and structural analyses revealed marked species-specific inhibitor susceptibility among fungal sterylglucosidases despite highly conserved overall architecture and catalytic machinery. Structural modeling and docking studies identified differences in ligand positioning and active-site interactions that likely contribute to these divergent pharmacological responses. Functionally, loss of SGL1 significantly attenuated virulence in a murine model of systemic candidiasis. Importantly, vaccination with heat-killed Δ sgl 1 cells conferred 70% protection against lethal systemic challenge and markedly reduced kidney fungal burden, providing the first evidence that sterylglucosidase-dependent vaccination can induce protective immunity against a non-pulmonary fungal infection. Collectively, these findings establish C. albicans Sgl1 as a conserved regulator of sterylglucoside homeostasis, fungal pathogenicity, and host protection, and identify fungal sterylglucosidases as promising targets for antifungal drug and immunotherapy development. IMPORTANCE Fungal infections are associated with substantial morbidity and mortality, yet effective immunotherapies and novel antifungal targets remain limited. Candida albicans is a major cause of invasive and mucosal fungal disease, highlighting the need for new therapeutic strategies. Here, we identify the fungus-specific sterylglucosidase Sgl1 as a conserved regulator of ergosterol 3-β-D-glucoside homeostasis and a critical determinant of C. albicans pathogenicity. We further demonstrate that fungal sterylglucosidases exhibit distinct species-specific pharmacological responses despite conserved structural frameworks, revealing opportunities for rational antifungal drug design. Importantly, deletion of SGL1 attenuated virulence and enabled protective vaccination against lethal systemic candidiasis, providing the first evidence that targeting sterylglucoside metabolism can elicit protective immunity against a non-pulmonary fungal pathogen. Together, these findings identify Candida albicans Sgl1 as a promising target for antifungal drug and immunotherapy development.
ABSTRACT Coronaviruses continue to pose a global public health threat, and the increasing frequency of zoonotic spillover events highlights the crucial role of animal hosts in cross-species transmission. As companion animals, cats warrant attention for their potential involvement in coronavirus cross-species spread. Here, we show that feline aminopeptidase N (fAPN) functions as a shared receptor utilized by multiple alphacoronaviruses (α-CoVs), including human coronavirus 229E (HCoV-229E), transmissible gastroenteritis virus, and type-II feline infectious peritonitis virus, to mediate entry into feline cells. Structural and functional analyses reveal that distinct viruses engage fAPN through alternative binding sites, enabling multimodal receptor recognition, and demonstrating notable receptor compatibility. Animal challenge experiments further indicate that HCoV-229E showed detectable viral RNA and infectious virus in the feline respiratory tract, with no apparent clinical signs. Together, these findings establish fAPN as a shared receptor for diverse α-CoVs and reveal a molecular basis for receptor compatibility across viral lineages, providing insight into the determinants of coronavirus host range and cross-species transmission. IMPORTANCE Zoonotic coronaviruses repeatedly threaten human health, yet the rules governing their ability to cross species remain incompletely understood. Cats live in close contact with humans and may act as hidden hosts for coronaviruses, but the molecular basis supporting cross-species infection in these animals is unclear. This study demonstrates that feline aminopeptidase N (fAPN) serves as a common receptor for several alphacoronaviruses (α-CoVs), including human coronavirus 229E (HCoV-229E), transmissible gastroenteritis virus, and type-II feline infectious peritonitis virus. These viruses recognize fAPN through distinct binding sites, highlighting the flexibility of receptor recognition. Additionally, HCoV-229E inoculation resulted in detectable viral RNA in the respiratory tract of cats, without apparent clinical signs. These results help explain why α-CoVs can move across host barriers and suggest that cats should be considered when assessing coronavirus spillover risks at the human-animal interface.
ABSTRACT The cefazolin inoculum effect (CzIE) is a clinically relevant in vitro phenomenon that may compromise cefazolin efficacy in high-inoculum methicillin-susceptible Staphylococcus aureus (MSSA) infections such as bacteremia and infective endocarditis. We aimed to define mechanisms underlying CzIE beyond BlaZ-dependent enzymatic pathways. We evaluated CzIE in 198 MSSA isolates (single center) by comparing minimum inhibitory concentrations at standard and high inoculum. The inoculum effect index was used as a quantitative measure of the CzIE to validate the operational definition and assess its concordance with the classical definition of CzIE. Whole-genome sequencing was used to define β-lactamase gene ( blaZ ) allotypes, accessory gene regulator ( agr) genotypes (I–IV), and clonal lineages. CzIE was observed in 16.16% of isolates, with Type A (71.9%) and Type C (28.1%) blaZ allotypes predominating among CzIE-positive strains. These isolates were enriched within clonal complex CC8 and CC30. Twenty-four-hour time-kill experiments combining cefazolin with BlaZ inhibitor (sulbactam) demonstrated initial killing followed by regrowth in CzIE-positive isolates, while complete eradication was observed in CzIE-negative isolates. Population analysis profiles (PAP) revealed the expansion of cefazolin–non-susceptible subpopulations at high inoculum, consistent with phenotypic heterogeneity. agr typing identified agr III as the predominant genotype among CzIE-positive isolates, and agr dysfunction was significantly associated with CzIE-positive phenotype. Together, these findings suggest that CzIE is driven not only by BlaZ-mediated activity but also by agr -dependent regulatory pathways. These observations highlight the potential clinical importance of CzIE and support further investigation into its implications for therapeutic optimization and improved patient outcomes. IMPORTANCE Cefazolin inoculum effect (CzIE) represents an underrecognized potential determinant of therapeutic failure in methicillin-susceptible Staphylococcus aureus (MSSA) infections, yet it remains absent from routine susceptibility testing frameworks. We demonstrate that CzIE is not solely driven by blaZ-mediated activity but is strongly associated with agr-dependent regulation and population heterogeneity. Our findings suggest that standard MIC testing can underestimate cefazolin failure under high-inoculum conditions commonly encountered in invasive diseases. Importantly, identification of CzIE-positive isolates could inform therapeutic decision-making, including selection of alternative β-lactams or adjunctive strategies to suppress tolerant subpopulations. Integration of CzIE assessment with genomic markers such as blaZ allotype and agr genotype may enable more precise prediction of treatment response. These data provide a framework for incorporating CzIE into diagnostic workflows and guide future studies evaluating its clinical utility. Ultimately, recognizing CzIE as a multifactorial phenotype may improve antibiotic selection, reduce treatment failure, and enhance outcomes in serious MSSA infections across patient populations.
ABSTRACT Group B Streptococcus (GBS) colonizes the gastrointestinal (GI) and female reproductive tracts of up to 30% of healthy adults, but during pregnancy can be transmitted to the newborn, causing neonatal sepsis, pneumonia, and meningitis. Neonatal GI colonization is an important precursor to invasive disease, yet the mechanisms governing GBS intestinal persistence and dissemination are still being elucidated. Given the predominance of hypervirulent sequence type 17 (ST-17) strains in neonatal invasive disease, we hypothesized that the ST-17-associated surface adhesin BspC contributes to gastrointestinal colonization and infection. Using a neonatal mouse model, we found that pups infected with wild-type GBS exhibited increased mortality and significantly higher bacterial burdens in the intestine, blood, spleen, and brain compared with pups infected with a Δ bspC mutant. We also observed that BspC promoted adherence to a 2D organoid model of the human infant colon and Caco-2 intestinal epithelial cells through its variable domain. Building on previous findings that Candida albicans interacts with BspC, we found that C. albicans enhanced GBS intestinal epithelial adherence in a BspC-dependent manner, and coinfection increased intestinal GBS burden and brain dissemination in neonatal mice. Finally, analysis of published human neonatal fecal microbiome data sets revealed higher GBS abundance in infants colonized with C. albicans or other Candida species. Together, these findings identify BspC as a key mediator of GBS intestinal colonization and invasive disease and suggest that fungal colonization of the neonatal GI promotes GBS pathogenesis. IMPORTANCE Group B Streptococcus (GBS) is a leading cause of neonatal infection, which can result in fatality or long-term neurological morbidity in survivors. Intestinal colonization is an important precursor to invasive disease, yet the factors that promote GBS persistence in the neonatal gastrointestinal tract remain incompletely understood. Here, we identify the GBS surface adhesin BspC as a mediator of intestinal epithelial adherence, gastrointestinal colonization, and systemic dissemination. We further demonstrate that the common fungal pathobiont Candida albicans enhances GBS intestinal adherence and infection. Consistent with our experimental findings, analysis of human infant microbiome data sets revealed greater GBS abundance in infants colonized with Candida species. These findings identify BspC as a key mediator of this process and suggest that fungal colonization can enhance GBS disease.
