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To gain a full understanding of how bacteria infect tissues, it is necessary to characterize both the bacteria and the tissue at the time of infection. However, this analysis is very complex because it involves obtaining infected tissue directly from patients and analyzing it with minimal processing to preserve the characteristics of the natural state of infection. In this study, we examined the gene expression profiles of replaced heart valves from patients with Staphylococcus aureus infection and compared them with pig valves experimentally infected with the same bacteria. Our findings provide a detailed insight into the changes occurring in the infected tissue and the bacterial adaptations required for multiplication and survival on the valve tissue. Notably, the strong similarity observed between human and porcine valves confirms that the porcine endocarditis model closely mirrors the human condition, making it a valuable tool for testing new therapies against this serious infection.

Infections with Staphylococcus aureus pose a serious public health threat due to high levels of antibiotic resistance and limited efficacy of alternative therapeutics. There has been a great deal of interest in developing novel therapeutics that target virulence factors essential during infection. However, it remains largely unknown if these factors are required at specific stages of the infection, and whether all bacterial cells or a limited subset express them. Here, we sought to examine virulence factor expression using fluorescent reporter strains that would indicate activity of two master regulators of virulence in S. aureus , Agr and Sae. While Agr appeared inactive during kidney abscess development, the Sae system exhibited heterogeneity, increased expression at later stages, and was required for abscess progression. These results provide critical information for the development of virulence factor-targeting strategies for kidney abscess treatment.

is one of the most common fungal pathogens, yet much remains unknown about how its virulence factors cooperate to promote pathogenicity. To investigate this, CRISPR-Cas9 technology was used to create a panel of 19 single, double, triple, and quadruple deletion mutant strains targeting four established virulence factors: (adhesin/invasin), (candidalysin toxin), (hypha formation regulator), and (protease). , the deletion of each gene had differing impacts across multiple characterization assays. The ∆/∆ mutant was unable to form hyphae under inducing conditions, leading to downstream impairment of epithelial invasion. The ∆/∆ mutant exhibited significantly reduced adhesion and invasion into epithelial cells, resulting in attenuated cellular damage. The ∆/∆ mutant displayed significantly reduced epithelial damage, cell signaling, and immune activation. The phenotype of the ∆/∆ mutant resembled that of wild type but was unable to degrade protein. In an immunocompromised murine model of oropharyngeal infection, hyphal growth and candidalysin production were the dominant drivers of elevated fungal burden, innate immune responses, and mortality. Following a 5-day infection with ∆/∆ and ∆/∆ single gene deletion strains, mice had survival rates of 100% and 80%, respectively, compared to 15% in wild-type infected mice. Notably, 100% survival was also observed following challenge with all ∆/∆ and ∆/∆ combination mutants. This study demonstrates that specific virulence attributes act in combination to promote mucosal infection, with hyphal growth and candidalysin production being a critical driver of oropharyngeal infection.IMPORTANCE has been classified by the WHO as a \"critical priority\" pathogen, highlighting the urgent need for a greater understanding of the mechanisms that enable it to cause disease. possesses numerous virulence attributes, but how they synergize during infection is not well understood. Here, using reverse genetics, we dissect the individual and combinatorial roles of four virulence factors (Als3p, candidalysin, hyphal growth, and Sap2p) and in an murine model of oropharyngeal candidiasis. Increasing the number of gene deletions correlated with reduced oral fungal burden, with hyphal growth and candidalysin together being critical for infection, inflammation, and mortality during oropharyngeal infection. These findings demonstrate that virulence attributes act cooperatively as a collective network to promote pathogenicity, a finding also observed in plant fungal pathogens. Our approach has identified specific fungal virulence factors that can be targeted for new treatment strategies against infections.

Macrophages are one of the key target cells of pathogenic Yersinia , where central immune response pathways, such as phagocytosis, gene expression, and inflammasome assembly, are suppressed by secreted bacterial effectors (Yops) in a highly coordinated fashion. Most studies analyzing cooperation between Yop proteins have utilized cell lines and mouse-derived macrophages, which strongly differ from human macrophages. This study employed primary human macrophages and analyzed cooperation between different Yersinia enterocolitica effector proteins on gene expression, histone phosphorylation, calcium signaling, and inflammasome assembly. We reveal synergistic, antagonistic, and individual roles of different Yersinia effector proteins. This work highlights how highly coordinated activities of a limited set of effectors can efficiently disarm macrophage immune responses and lead to a successful infection.

For bacterial pathogens to establish infection and persist in the host, they must adapt to harsh environments and fine-tune gene expression accordingly. The transcriptional antiterminator RfaH plays a pivotal role in regulating key genes essential for adaptation and virulence in Y. pseudotuberculosis . In this study, we explored the function of RfaH in bacterial physiology, stress responses, and infection dynamics. Using a mouse infection model, we found that loss of RfaH significantly reduced virulence and impaired the pathogen’s ability to establish persistent infection. Notably, RfaH expression increased under stress conditions, such as high osmolarity and temperature, underscoring its role in bacterial adaptation. On the other hand, the absence of RfaH led to motility defects and enhanced bacterial aggregation, suggesting alterations in surface properties. Transcriptomic analysis revealed that RfaH influences a broader set of genes beyond the O-antigen biosynthesis operon, including virulence factors critical for host adaptation. Overall, our findings establish RfaH as a key regulator of Y. pseudotuberculosis virulence, shedding light on the molecular mechanisms that enable bacterial survival in challenging environments.

Kingella kingae is an emerging pediatric pathogen and a leading cause of osteoarticular infections in children 6 months to 4 years of age. To establish infection, K. kingae relies on T4P, dynamic surface structures that mediate host cell adherence, motility, and DNA uptake. T4P are expressed by a wide range of bacterial pathogens beyond K. kingae , including Pseudomonas aeruginosa , Neisseria gonorrhoeae , Neisseria meningitidis , and Legionella pneumophila , among others. The type IV pilus is composed of pilin subunits, including a major pilin that displays significant antigenic diversity and low-abundance minor pilins that are highly conserved. This study demonstrates the importance of eight minor pilins in K. kingae virulence properties. Given the conservation of minor pilins across diverse bacterial species, targeting minor pilin complexes may provide a foundation for a new class of broad-spectrum antivirulence therapies that prevent bacterial colonization and disease.

During infection processes, pathogens cope with host-mediated stressors. In response to those stressors, bacteria adapt their gene expression as well as their proteome profile. In the pathogen Staphylococcus aureus , protein homeostasis is mainly controlled by the Clp system. In particular, ClpX is the most conserved Clp unfoldase and is involved in the overall regulation of virulence and bacterial fitness. However, the majority of ClpX targets remain elusive in S. aureus . With our proteomics approach and in-depth data analysis, we provide a resource for global insight into ClpX-dependent adaptation of S. aureus physiology under infection-relevant conditions. Based on this, we uncover ClpX’s role as a central player in the iron and oxygen limitation response. In addition, we demonstrate the importance of ClpX in S. aureus bacterial fitness in infection processes. However, reduced levels of ClpX lead to high intracellular persistence, which questions ClpX’s suitability as a therapeutic target.

is a growing public health concern, capable of causing long-term contamination of healthcare settings, skin colonization, and life-threatening bloodstream infections. However, pathogenesis is not well understood, which is exacerbated by limitations and discrepancies in existing animal infection models. Further, the effects of growth phase on virulence have not been examined, despite growth phase being linked to virulence in many bacterial species. To address this question, and to develop an immunocompetent murine model of infection, we directly compared log-phase and stationary-phase systemic infection in immunocompetent C57BL/6J mice at high and low doses of infection. Systemic infection with high-dose log-phase results in rapid mortality between 2 h and 1 day post-infection, whereas stationary phase results in significantly extended survival. However, at low doses of infection, there was no difference in mortality kinetics between log-phase and stationary-phase cells. We observed that initially colonizes multiple organs but is rapidly cleared from the lungs and spleen, while kidney fungal burdens remain stable. Mice infected with high-dose log-phase had fibrin-associated blood clotting in multiple organs and decreased serum fibrinogen levels, suggesting that coagulation may drive rapid mortality. This was associated with increased β-glucan exposure and mannan abundance in log-phase . These results will inform the development of a more standardized animal model of systemic infection, which can be used to reveal key aspects of pathogenesis.IMPORTANCEDespite its growing medical importance, there is limited understanding of pathogenesis, due in part to limitations of existing laboratory models of infection. To develop a more complete understanding of factors that contribute to pathogenesis, it will be necessary to establish consistent parameters for animal models of infection. To address this need, we directly compared log and stationary growth phases on pathogenesis in immunocompetent C57BL/6J mice using a single virulent Clade I isolate. At a high dose of infection, host survival was dramatically different between log-phase or stationary-phase , suggesting that growth phase can affect pathogenesis. These differences correlated with increased exposure of pathogen-associated molecular patterns in the cell wall in log-phase cells. These results will be instrumental in the future development of standardized animal models to study pathogenesis.

Edwardsiella piscicida causes severe hemorrhagic septicemia in marine and freshwater fish worldwide, resulting in significant economic losses for the aquaculture industry (K. Y. Leung, Q. Wang, Z. Yang, and B. A. Siame, Virulence 10:555–567, 2019, https://doi.org/10.1080/21505594.2019.1621648 ). Our previous research identified a novel type III secretion system effector, EseQ, in E. piscicida whose function remains to be elucidated. In this work, we showed that EseQ binds to tubulin and GEF-H1 and destabilizes microtubules. GEF-H1 released from microtubules activates the RhoA-ROCK-MLCII signaling pathway, leading to stress fiber formation in epithelial cells. EseQ deforms the epithelial barrier and promotes E. piscicida ’s invasion in a stress fiber-dependent manner. This work contributes to the understanding of the mechanism by which E. piscicida invades host cells.

This study reveals R. callidus as a key gut microbiota species enriched in CRC patients with high CEA levels, demonstrating its novel pro-tumor associations through positive correlations with mast cell infiltration and CXCL1 chemokine and upregulation of long-chain fatty acid metabolism. Concurrently, we identify distinct immune micro-environments: elevated resting memory CD4+ T cells in high-CEA patients versus increased T follicular helper cells in low-CEA cohorts. Critically, by leveraging 30 differential microbial features, we develop ML models for noninvasive prediction of CEA levels. These findings establish gut microbiota as both a mechanistic mediator of CEA-driven CRC progression and a foundation for microbiome-based diagnostic tools.