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Aim The aim of this study was to assess the clinical significance and prognostic value of the preoperative fibrinogen (FBG) level in patients with native valve infective endocarditis (NVIE) who underwent valve surgery. Methods This retrospective study included a total of 163 consecutive patients who were diagnosed with NVIE and underwent valve surgery from January 2019 to January 2022 in our hospital. The primary endpoint was all-cause mortality. Results All-cause mortality was observed in 9.2% of the patients (n = 15). Body mass index (BMI) was lower in the survival group (p = 0.025), whereas FBG (p = 0.008) and platelet count (p = 0.044) were significantly greater in the survival group than in the death group. Multivariate Cox proportional hazards analysis revealed that FBG (HR, 0.55; 95% CI, [0.32–0.94]; p = 0.029) was an independent prognostic factor for all-cause mortality. Furthermore, Kaplan‒Meier survival curve analysis revealed that patients with low FBG levels (3.99 g/L). In the trend analysis, the FBG tertiles were significantly related to all-cause mortality in all three adjusted models, and the p values for trend were 0.017, 0.016, and 0.028, respectively. Conclusion Preoperative FBG may serve as a prognostic factor for all-cause mortality, and an FBG concentration less than 3.28 g/L was associated with a greater risk of all-cause mortality in NVIE patients undergoing valve surgery.

Abstract Membrane vesicles (MVs) are produced by species across all domains of life and have diverse physiological functions as well as promising applications. While the mechanisms for vesiculation in Gram‐negative bacteria are well‐established, the genetic determinants and regulatory factors responsible for MV biogenesis in Gram‐positive bacteria remain largely unknown. Here, we demonstrate that a Q225P substitution in the alternative sigma factor B (SigB) triggers MV production in Staphylococcus aureus strain Newman by hindering the specific binding of SigB to the asp23 promoter, thereby repressing expression of alkaline shock protein 23 (Asp23). Isogenic deletion of asp23 also promotes MV formation in Newman, confirming the critical roles played by sigB and asp23 in modulating S. aureus vesiculation. While bacterial growth and cytoplasmic membrane fluidity are not impaired, mutation of asp23 weakens the cell wall and enhances autolysis, consistent with decreased expression of alpha‐type psm and lrgAB that modulate murein hydrolase activity. TEM and proteomic analysis show that Newman and asp23 deletion mutant generate MVs with nearly identical morphology and composition, but virulence‐associated factors are significantly enriched in MVs from the asp23 mutant. Overall, this study reveals novel genetic determinants underlying S. aureus vesiculation and advances the understanding of the physiology of MV biogenesis in S. aureus.

ABSTRACT The underlying mechanism of thermotolerance, which is a key virulence factor essential for pathogenic fungi such as Cryptococcus neoformans, is largely unexplored. In this study, our findings suggest that Set302, a homolog of Set3 and a subunit of histone deacetylase complex Set3C, contributes to thermotolerance in C. neoformans. Specifically, the deletion of the predicted Set3C core subunit, Set302, resulted in further reduction in the growth of C. neoformans at 39°C, and survival of transient incubation at 50°C. Transcriptomics analysis revealed that the expression levels of numerous heat stress-responsive genes altered at both 30°C and 39°C due to the lack of Set302. Notably, at 39°C, the absence of Set302 led to the downregulation of gene expression related to the ubiquitin-proteasome system (UPS). Based on the GFP-α-synuclein overexpression model to characterize misfolded proteins, we observed a pronounced accumulation of misfolded GFP-α-synuclein at 39°C, consequently inhibiting C. neoformans thermotolerance. Furthermore, the loss of Set302 exacerbated the accumulation of misfolded GFP-α-synuclein during heat stress. Interestingly, the set302∆ strain exhibited a similar phenotype under proteasome stress as it did at 39°C. Moreover, the absence of Set302 led to reduced production of capsule and melanin. set302∆ strain also displayed significantly reduced pathogenicity and colonization ability compared to the wild-type strain in the murine infection model. Collectively, our findings suggest that Set302 modulates thermotolerance by affecting the degradation of misfolded proteins and multiple virulence factors to mediate the pathogenicity of C. neoformans.IMPORTANCECryptococcus neoformans is a pathogenic fungus that poses a potential and significant threat to public health. Thermotolerance plays a crucial role in the wide distribution in natural environments and host colonization of this fungus. Herein, Set302, a critical core subunit for the integrity of histone deacetylase complex Set3C and widely distributed in various fungi and mammals, governs thermotolerance and affects survival at extreme temperatures as well as the formation of capsule and melanin in C. neoformans. Additionally, Set302 participates in regulating the expression of multiple genes associated with the ubiquitin-proteasome system (UPS). By eliminating misfolded proteins under heat stress, Set302 significantly contributes to the thermotolerance of C. neoformans. Moreover, Set302 regulates the pathogenicity and colonization ability of C. neoformans in a murine model. Overall, this study provides new insight into the mechanism of thermotolerance in C. neoformans.

Burkholderia pseudomallei is the causal agent of melioidosis, a deadly tropical infectious disease that lacks a vaccine. On the basis of the attenuated Staphylococcus aureus RN4220-Δagr (RN), we engineered the RN4220-Δagr/pdhB-hcp1 strain (RN-Hcp1) to generate B. pseudomallei hemolysin-coregulated protein 1 (Hcp1)-loaded membrane vesicles (hcp1MVs). The immunization of BALB/c mice with hcp1MVs mixed with adjuvant by a three-dose regimen increased the serum specific IgG production. The serum levels of inflammatory factors, including TNF-α and IL-6, in hcp1MV-vaccinated mice were comparable with those in PBS-challenged mice. The partial adjuvant effect of staphylococcal MVs was observed with the elevation of specific antibody titer in hcp1MV-vaccinated mice relative to those that received the recombinant Hcp1 protein (rHcp1) or MVs derived from RN strain (ΔagrMVs). The hcp1MVs/adjuvant vaccine protected 70% of mice from lethal B. pseudomallei challenge. Immunization with hcp1MVs only protected 60% of mice, whereas vaccination with rHcp1 or ΔagrMVs conferred no protection. Moreover, mice that received hcp1MVs/adjuvant and hcp1MVs immunization had low serum TNF-α and IL-6 levels and no inflammatory infiltration in comparison with other groups. In addition, all surviving mice in hcp1MVs/adjuvant and hcp1MVs groups exhibited no culturable bacteria in their lungs, livers, and spleens five days postinfection. Overall, our data highlighted a new strategy for developing B. pseudomallei vaccine and showed that Hcp1-incorporated staphylococcal MV is a promising candidate for the prevention of acute melioidosis.

ABSTRACT Staphylococcus aureus represents a major human pathogen that is frequently involved in polymicrobial infections. However, the prevalence and role of co-infectious microbes on the pathogenesis and fitness essentiality of S. aureus in vivo remain largely unknown. In this study, we firstly performed a retrospective surveillance of 760 clinical samples and revealed a notable predominance of co-infection with S. aureus and Acinetobacter baumannii. The high-density S. aureus transposon mutant library coupled to transposon insertion sequencing (Tn-Seq) further identified a core set of genes enriched in metabolism of inorganic ions, amino acids, and carbohydrates, which are essential for infection and tissue colonization of S. aureus in the murine systemic infection model. Notably, we revealed a differential requirement of fitness factors for S. aureus in tissue-specific (liver and kidney) and infection-type-specific manner (mono- and co-infection). Co-infection with A. baumannii dramatically altered the fitness requirements of S. aureus in vivo; 49% of the mono-infection fitness genes in S. aureus strain Newman were converted to non-essential, and the functionality of ATP-binding cassette (ABC) transporters was significantly elicited during co-infection. Furthermore, the number of genes essential during co-infection (503) outnumbers the genes essential during mono-infection (362). In addition, the roles of 3 infection-type-specific genes in S. aureus during mono-infection or co-infection with A. baumannii were validated with competitive experiments in vivo. Our data indicated a high incidence and clinical relevance of S. aureus and A. baumannii co-infection, and provided novel insights into establishing antimicrobial regimens to control co-infections. IMPORTANCE Polymicrobial infections are widespread in clinical settings, which potentially correlate with increased infection severity and poor clinical outcomes. Staphylococcus aureus is a formidable human pathogen that causes a variety of diseases in polymicrobial nature. Co-infection and interaction of S. aureus have been described with limited pathogens, mainly including Pseudomonas aeruginosa, Candida albicans, and influenza A virus. Thus far, the prevalence and role of co-infectious microbes on the pathogenesis and fitness essentiality of S. aureus in vivo remain largely unknown. Understanding the polymicrobial composition and interaction, from a community and genome-wide perspective, is thus crucial to shed light on S. aureus pathogenesis strategy. Here, our findings demonstrated, for the first time, that a high incidence rate and clinical relevance of co-infection was caused by S. aureus and Acinetobacter baumannii, illustrating the importance of polymicrobial nature in investigating S. aureus pathogenesis. The infection-type-specific genes likely serve as potential therapeutic targets to control S. aureus infections, either in mono- or co-infection situation, providing novel insights into the development of antimicrobial regimens to control co-infections.

Bacterial membrane vesicles (MVs) are produced by both Gram-positive and Gram-negative bacteria during growth in vitro and in vivo. MVs are nanoscale vesicular structures with diameters ranging from 20 to 400 nm. MVs incorporate bacterial lipids, proteins, and often nucleic acids, and can effectively stimulate host immune response against bacterial infections. As vaccine candidates and drug delivery systems, MVs possess high biosafety owing to the lack of self-replication ability. However, wild-type bacterial strains have poor MV yield, and MVs from the wild-type strains may be harmful due to the carriage of toxic components, such as lipopolysaccharides, hemolysins, enzymes, etc. In this review, we summarize the genetic modification of vesicle-producing bacteria to reduce MV toxicity, enhance vesicle immunogenicity, and increase vesicle production. The engineered MVs exhibit broad applications in vaccine designs, vaccine delivery vesicles, and drug delivery systems.

Staphylococcus aureus represents a notorious opportunistic pathogen causing various infections in biofilm nature, imposing remarkable therapeutic challenges worldwide. The catabolite control protein A (CcpA), a major regulator of carbon catabolite repression (CCR), has been recognized to modulate S. aureus biofilm formation, while the underlying mechanism remains to be fully elucidated. In this study, the reduced biofilm was firstly determined in the ccpA deletion mutant of S. aureus clinical isolate XN108 using both crystal violet staining and confocal laser scanning microscopy. RNA-seq analysis suggested that sak-encoding staphylokinase (Sak) was significantly upregulated in the mutant ∆ccpA, which was further confirmed by RT-qPCR. Consistently, the induced Sak production correlated the elevated promoter activity of sak and increased secretion in the supernatants, as demonstrated by Psak-lacZ reporter fusion expression and chromogenic detection, respectively. Notably, electrophoretic mobility shift assays showed that purified recombinant protein CcpA binds directly to the promoter region of sak, suggesting the direct negative control of sak expression by CcpA. Double isogenic deletion of ccpA and sak restored biofilm formation for mutant ∆ccpA, which could be diminished by trans-complemented sak. Furthermore, the exogenous addition of recombinant Sak inhibited biofilm formation for XN108 in a dose-dependent manner. Together, this study delineates a novel model of CcpA-controlled S. aureus biofilm through direct inhibition of sak expression, highlighting the multifaceted roles and multiple networks regulated by CcpA.