Mouse Model Of Systemic Infection Research Articles

The hinge-engineered IgG1-IgG3 hybrid subclass IgGh47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies

Streptococcus pyogenes can cause invasive disease with high mortality despite adequate antibiotic treatments. To address this unmet need, we have previously generated an opsonic IgG1 monoclonal antibody, Ab25, targeting the bacterial M protein. Here, we engineer the IgG2-4 subclasses of Ab25. Despite having reduced binding, the IgG3 version promotes stronger phagocytosis of bacteria. Using atomic simulations, we show that IgG3’s Fc tail has extensive movement in 3D space due to its extended hinge region, possibly facilitating interactions with immune cells. We replaced the hinge of IgG1 with four different IgG3-hinge segment subclasses, IgGhxx. Hinge-engineering does not diminish binding as with IgG3 but enhances opsonic function, where a 47 amino acid hinge is comparable to IgG3 in function. IgGh47 shows improved protection against S. pyogenes in a systemic infection mouse model, suggesting that IgGh47 has promise as a preclinical therapeutic candidate. Importantly, the enhanced opsonic function of IgGh47 is generalizable to diverse S. pyogenes strains from clinical isolates. We generated IgGh47 versions of anti-SARS-CoV-2 mAbs to broaden the biological applicability, and these also exhibit strongly enhanced opsonic function compared to the IgG1 subclass. The improved function of the IgGh47 subclass in two distant biological systems provides new insights into antibody function.

The Putative Cytochrome b5 Domain-Containing Protein CaDap1 Homologue Is Involved in Antifungal Drug Tolerance, Cell Wall Chitin Maintenance, and Virulence in Candida albicans.

Candida albicans (Ca), a prominent opportunistic fungal pathogen in humans, has garnered considerable attention due to its infectious properties. Herein, we have identified and characterized CaCDAP1 (Ca orf19.1034), a homolog of ScDAP1 found in Saccharomyces cerevisiae. CaCDAP1 encodes a 183-amino acid protein with a conserved cytochrome b5-like heme-binding domain. The deletion of CaDAP1 renders Ca cells susceptible to caspofungin and terbinafine. CaDAP1 deletion confers resistance to Congo Red and Calcofluor White, and sensitivity to sodium dodecyl sulfate. The deletion of CaDAP1 results in a 50% reduction in chitin content within the cell wall, the downregulation of phosphorylation levels in CaMkc1, and the upregulation of phosphorylation levels in CaCek1. Notably, CaDAP1 deletion results in the abnormal hyphal development of Ca cells and diminishes virulence in a mouse systemic infection model. Thus, CaDAP1 emerges as a critical regulator governing cellular responses to antifungal drugs, the synthesis of cell wall chitin, and virulence in Ca.

Antibacterial activity of the antimicrobial peptide PMAP-36 in combination with tetracycline against porcine extraintestinal pathogenic Escherichia coli in vitro and in vivo

The increase in the emergence of antimicrobial resistance has led to great challenges in controlling porcine extraintestinal pathogenic Escherichia coli (ExPEC) infections. Combinations of antimicrobial peptides (AMPs) and antibiotics can synergistically improve antimicrobial efficacy and reduce bacterial resistance. In this study, we investigated the antibacterial activity of porcine myeloid antimicrobial peptide 36 (PMAP-36) in combination with tetracycline against porcine ExPEC PCN033 both in vitro and in vivo. The minimum bactericidal concentrations (MBCs) of AMPs (PMAP-36 and PR-39) against the ExPEC strains PCN033 and RS218 were 10 μM and 5 μM, respectively. Results of the checkerboard assay and the time-kill assay showed that PMAP-36 and antibiotics (tetracycline and gentamicin) had synergistic bactericidal effects against PCN033. PMAP-36 and tetracycline in combination led to PCN033 cell wall shrinkage, as was shown by scanning electron microscopy. Furthermore, PMAP-36 delayed the emergence of PCN033 resistance to tetracycline by inhibiting the expression of the tetracycline resistance gene tetB. In a mouse model of systemic infection of PCN033, treatment with PMAP-36 combined with tetracycline significantly increased the survival rate, reduced the bacterial load and dampened the inflammatory response in mice. In addition, detection of immune cells in the peritoneal lavage fluid using flow cytometry revealed that the combination of PMAP-36 and tetracycline promoted the migration of monocytes/macrophages to the infection site. Our results suggest that AMPs in combination with antibiotics may provide more therapeutic options against multidrug-resistant porcine ExPEC.

Lipoteichoic acids influence cell shape and bacterial division of Streptococcus suis serotype 2, but play a limited role in the pathogenesis of the infection

Streptococcus suis serotype 2 is a major swine pathogen and a zoonotic agent, causing meningitis in both swine and humans, responsible for substantial economic losses to the swine industry worldwide. The pathogenesis of infection and the role of bacterial cell wall components in virulence have not been fully elucidated. Lipoproteins, peptidoglycan, as well as lipoteichoic acids (LTA) have all been proposed to contribute to virulence. In the present study, the role of the LTA in the pathogenesis of the infection was evaluated through the characterisation of a mutant of the S. suis serotype 2 strain P1/7 lacking the LtaS enzyme, which mediates the polymerization of the LTA poly-glycerolphosphate chain. The ltaS mutant was confirmed to completely lack LTA and displayed significant morphological defects. Although the bacterial growth of this mutant was not affected, further results showed that LTA is involved in maintaining S. suis bacterial fitness. However, its role in the pathogenesis of the infection appears limited. Indeed, LTA presence reduces self-agglutination, biofilm formation and even dendritic cell activation, which are important aspects of the pathogenesis of the infection caused by S. suis. In addition, it does not seem to play a critical role in virulence using a systemic mouse model of infection.

Evaluation of outbreak persistence caused by multidrug-resistant and echinocandin-resistant Candida parapsilosis using multidimensional experimental and epidemiological approaches

ABSTRACT Candida parapsilosis is known to cause severe and persistent outbreaks in clinical settings. Patients infected with multidrug-resistant C. parapsilosis (MDR Cp) isolates were identified in a large Turkish hospital from 2017–2020. We subsequently identified three additional patients infected with MDR Cp isolates in 2022 from the same hospital and two echinocandin-resistant (ECR) isolates from a single patient in another hospital. The increasing number of MDR and ECR isolates contradicts the general principle that the severe fitness cost associated with these phenotypes could prevent their dominance in clinical settings. Here, we employed a multidimensional approach to systematically assess the fitness costs of MDR and ECR C. parapsilosis isolates. Whole-genome sequencing revealed a novel MDR genotype infecting two patients in 2022. Despite severe in vitro defects, the levels and tolerances of the biofilms of our ECR and MDR isolates were generally comparable to those of susceptible wild-type isolates. Surprisingly, the MDR and ECR isolates showed major alterations in their cell wall components, and some of the MDR isolates consistently displayed increased tolerance to the fungicidal activities of primary human neutrophils and were more immunoevasive during exposure to primary human macrophages. Our systemic infection mouse model showed that MDR and ECR C. parapsilosis isolates had comparable fungal burden in most organs relative to susceptible isolates. Overall, we observed a notable increase in the genotypic diversity and frequency of MDR isolates and identified MDR and ECR isolates potentially capable of causing persistent outbreaks in the future.

The C-terminal protein interaction domain of the chromatin reader Yaf9 is critical for pathogenesis of Candida albicans.

Fungal infections cause a large health burden but are treated by only a handful of antifungal drug classes. Chromatin factors have emerged as possible targets for new antifungals. These targets include the reader proteins, which interact with posttranslationally modified histones to influence DNA transcription and repair. The YEATS domain is one such reader recognizing both crotonylated and acetylated histones. Here, we performed a detailed structure/function analysis of the Candida albicans YEATS domain reader Yaf9, a subunit of the NuA4 histone acetyltransferase and the SWR1 chromatin remodeling complex. We have previously demonstrated that the homozygous deletion mutant yaf9Δ/Δ displays growth defects and is avirulent in mice. Here we show that a YEATS domain mutant expected to inactivate Yaf9's chromatin binding does not display strong phenotypes in vitro, nor during infection of immune cells or in a mouse systemic infection model, with only a minor virulence reduction in vivo. In contrast to the YEATS domain mutation, deletion of the C-terminal domain of Yaf9, a protein-protein interaction module necessary for its interactions with SWR1 and NuA4, phenocopies the null mutant. This shows that the C-terminal domain is essential for Yaf9 roles in vitro and in vivo, including C. albicans virulence. Our study informs on the strategies for therapeutic targeting of Yaf9, showing that approaches taken for the mammalian YEATS domains by disrupting their chromatin binding might not be effective in C. albicans, and provides a foundation for studying YEATS proteins in human fungal pathogens.IMPORTANCEThe scarcity of available antifungal drugs and rising resistance demand the development of therapies with new modes of action. In this context, chromatin regulation may be a target for novel antifungal therapeutics. To realize this potential, we must better understand the roles of chromatin regulators in fungal pathogens. Toward this goal, here, we studied the YEATS domain chromatin reader Yaf9 in Candida albicans. Yaf9 uses the YEATS domain for chromatin binding and a C-terminal domain to interact with chromatin remodeling complexes. By constructing mutants in these domains and characterizing their phenotypes, our data indicate that the Yaf9 YEATS domain might not be a suitable therapeutic drug target. Instead, the Yaf9 C-terminal domain is critical for C. albicans virulence. Collectively, our study informs how a class of chromatin regulators performs their cellular and pathogenesis roles in C. albicans and reveals strategies to inhibit them.

MEndoB, a chimeric lysin featuring a novel domain architecture and superior activity for the treatment of staphylococcal infections.

Bacterial infections are a growing global healthcare concern, as an estimated annual 4.95 million deaths are associated with antimicrobial resistance (AMR). Methicillin-resistant Staphylococcus aureus is one of the deadliest pathogens and a high-priority pathogen according to the World Health Organization. Peptidoglycan hydrolases (PGHs) of phage origin have been postulated as a new class of antimicrobials for the treatment of bacterial infections, with a novel mechanism of action and no known resistances. The modular architecture of PGHs permits the creation of chimeric PGH libraries. In this study, the chimeric enzyme MEndoB was selected from a library of staphylococcal PGHs based on its rapid and sustained activity against staphylococci in human serum. The benefit of the presented screening approach was illustrated by the superiority of MEndoB in a head-to-head comparison with other PGHs intended for use against staphylococcal bacteremia. MEndoB displayed synergy with antibiotics and rapid killing in human whole blood with complete inhibition of re-growth over 24 h at low doses. Successful treatment of S. aureus-infected zebrafish larvae with MEndoB provided evidence for its in vivo effectiveness. This was further confirmed in a lethal systemic mouse infection model in which MEndoB significantly reduced S. aureus loads and tumor necrosis factor alpha levels in blood in a dose-dependent manner, which led to increased survival of the animals. Thus, the thorough lead candidate selection of MEndoB resulted in an outstanding second-generation PGH with in vitro, ex vivo, and in vivo results supporting further development.IMPORTANCEOne of the most pressing challenges of our era is the rising occurrence of bacteria that are resistant to antibiotics. Staphylococci are prominent pathogens in humans, which have developed multiple strategies to evade the effects of antibiotics. Infections caused by these bacteria have resulted in a high burden on the health care system and a significant loss of lives. In this study, we have successfully engineered lytic enzymes that exhibit an extraordinary ability to eradicate staphylococci. Our findings substantiate the importance of meticulous lead candidate selection to identify therapeutically promising peptidoglycan hydrolases with unprecedented activity. Hence, they offer a promising new avenue for treating staphylococcal infections.

Csu pili dependent biofilm formation and virulence of Acinetobacter baumannii

Acinetobacter baumannii has emerged as one of the most common extensive drug-resistant nosocomial bacterial pathogens. Not only can the bacteria survive in hospital settings for long periods, but they are also able to resist adverse conditions. However, underlying regulatory mechanisms that allow A. baumannii to cope with these conditions and mediate its virulence are poorly understood. Here, we show that bi-stable expression of the Csu pili, along with the production of poly-N-acetyl glucosamine, regulates the formation of Mountain-like biofilm-patches on glass surfaces to protect bacteria from the bactericidal effect of colistin. Csu pilus assembly is found to be an essential component of mature biofilms formed on glass surfaces and of pellicles. By using several microscopic techniques, we show that clinical isolates of A. baumannii carrying abundant Csu pili mediate adherence to epithelial cells. In addition, Csu pili suppressed surface-associated motility but enhanced colonization of bacteria into the lungs, spleen, and liver in a mouse model of systemic infection. The screening of c-di-GMP metabolizing protein mutants of A. baumannii 17978 for the capability to adhere to epithelial cells led us to identify GGDEF/EAL protein AIS_2337, here denoted PdeB, as a major regulator of Csu pili-mediated virulence and biofilm formation. Moreover, PdeB was found to be involved in the type IV pili-regulated robustness of surface-associated motility. Our findings suggest that the Csu pilus is not only a functional component of mature A. baumannii biofilms but also a major virulence factor promoting the initiation of disease progression by mediating bacterial adherence to epithelial cells.

Exploration and Biological Evaluation of 1,3-Diamino-7H-pyrrol[3,2-f]quinazoline Derivatives as Dihydrofolate Reductase Inhibitors.

Dihydrofolate reductase (DHFR), a core enzyme of folate metabolism, plays a crucial role in the biosynthesis of purines and thymidylate for cell proliferation and growth in both prokaryotic and eukaryotic cells. However, the development of new DHFR inhibitors is challenging due to the limited number of scaffolds available for drug development. Hence, we designed and synthesized a new class of DHFR inhibitors with a 1,3-diamino-7H-pyrrol[3,2-f]quinazoline derivative (PQD) structure bearing condensed rings. Compound 6r exhibited therapeutic effects on mouse models of systemic infection and thigh infection caused by methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300. Moreover, methyl-modified PQD compound 8a showed a strong efficacy in a murine model of breast cancer, which was better than the effects of taxol. The findings showcased in this study highlight the promising capabilities of novel DHFR inhibitors in addressing bacterial infections as well as breast cancer.

The virulence regulator AbsR in avian pathogenic Escherichia coli has pleiotropic effects on bacterial physiology

Avian pathogenic Escherichia coli (APEC) belonging to extraintestinal pathogenic E. coli (ExPEC) can cause severe infections in extraintestinal tissues in birds and humans, such as the lungs and blood. MprA (microcin production regulation, locus A, herein renamed AbsR, a blood survival regulator), a member of the MarR (multiple antibiotic resistance regulator) transcriptional regulator family, governs the expression of capsule biosynthetic genes in human ExPEC and represents a promising druggable target for antimicrobials. However, a deep understanding of the AbsR regulatory mechanism as well as its regulon is lacking. In this study, we present a systems-level analysis of the APEC AbsR regulon using ChIP-Seq (chromatin immunoprecipitation sequencing) and RNA-Seq (RNA sequencing) methods. We found that AbsR directly regulates 99 genes and indirectly regulates 667 genes. Furthermore, we showed that: 1) AbsR contributes to antiphagocytotic effects by macrophages and virulence in a mouse model for systemic infection by directly activating the capsular gene cluster; 2) AbsR positively impacts biofilm formation via direct regulation of the T2SS (type II secretion system) but plays a marginal role in virulence; and 3) AbsR directly upregulates the acid tolerance signaling system EvgAS to withstand acid stress but is dispensable in ExPEC virulence. Finally, our data indicate that the role of AbsR in virulence gene regulation is relatively conserved in ExPEC strains. Altogether, this study provides a comprehensive analysis of the AbsR regulon and regulatory mechanism, and our data suggest that AbsR likely influences virulence primarily through the control of capsule production. Interestingly, we found that AbsR severely represses the expression of the type I-F CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) systems, which could have implications in CRISPR biology and application.

Glucomannan as a polysaccharide adjuvant improved immune responses against Staphylococcus aureus: Potency and efficacy studies

Staphylococcus aureus is a gram-positive bacterium, representing one of the most important nosocomial pathogens. The treatment of infections, caused by S. aureus, has become increasingly intricate due to the emergence of highly resistant strains. Therefore, it is obvious that an effective prevention strategy against this bacterium could significantly decrease such infections. In the present study, the protective efficacy and immunological properties of recombinant autolysin, formulated in Montanide ISA266 and Alum adjuvants with Glucomannan as a polysaccharide, were assessed in the systemic mouse model of infection. Mice were immunized with the purified recombinant protein in various formulations in different groups and, subsequently, mice were challenged with 5 × 108 CFU of bacteria for the evaluation of their survival and bacterial clearances in the internal organs. ELISA was performed to determine the type of induced immunity, cytokine secretion (IFN-γ, IL-4, IL-2, and IL-17), and isotyping (IgG1 and IgG2a). In addition, we measured the opsonophagocytic activities of the antibodies.Results showed that immunization with r-autolysin + Alum + Glucomannan and r-autolysin + MontanideISA266+Glucomannan formulations significantly increased total IgG and isotypes (IgG1 and IgG2a), as compared with other vaccinated and control groups. Furthermore, the formulation of r-autolysin in Alum and MontanideISA266 adjuvants with Glucomannan enhanced IFN-γ, IL-4, and IL-17 cytokine secretion as well as protectivity, following experimental challenge.We concluded that Glucomannan has the potential to induce immune responses and would be used as an adjuvant factor in vaccine formulation.

The enzyme activity of sortase A is regulated by phosphorylation in Staphylococcus aureus

ABSTRACT In many Gram-positive bacteria, the transpeptidase enzyme sortase A (SrtA) anchors surface proteins to cell wall and plays a critical role in the bacterial pathogenesis. Here, we show that in Staphylococcus aureus, an important human pathogen, the SrtA is phosphorylated by serine/threonine protein kinase Stk1. S. aureus SrtA can also be phosphorylated by small-molecule phosphodonor acetyl phosphate (AcP) in vitro. We determined that various amino acid residues of S. aureus SrtA are subject to phosphorylation, primarily on its catalytic site residue cysteine-184 in the context of a bacterial cell lysate. Both Stk1 and AcP-mediated phosphorylation inhibited the enzyme activity of SrtA in vitro. Consequently, deletion of gene (i.e. stp1) encoding serine/threonine phosphatase Stp1, the corresponding phosphatase of Stk1, caused an increase in the phosphorylation level of SrtA. The stp1 deletion mutant mimicked the phenotypic traits of srtA deletion mutant (i.e. attenuated growth where either haemoglobin or haem as a sole iron source and reduced liver infections in a mouse model of systemic infection). Importantly, the phenotypic defects of the stp1 deletion mutant can be alleviated by overexpressing srtA. Taken together, our finding suggests that phosphorylation plays an important role in modulating the activity of SrtA in S. aureus.

In vitro and in vivo activities of a novel β-lactamase inhibitor combination imipenem/XNW4107 against recent clinical Gram-negative bacilli from China

XNW4107 is a novel β-lactamase inhibitor that possesses broad activity against serine-β-lactamases. XNW4107 in combination with imipenem exhibited potent in vitro activity against carbapenem-resistant bacteria and particularly against carbapenem-resistant Acinetobacter baumannii. This study aimed to evaluate the in vitro and in vivo antibacterial activities of imipenem/XNW4107. The minimum inhibitory concentrations, minimum bactericidal concentrations, time-kill curves, post-antibiotic effects, and spontaneous frequency of resistance were used to investigate the imipenem/XNW4107 in vitro activity. A mouse systemic infection model was used to evaluate the imipenem/XNW4107 in vivo efficacy. MIC90 of imipenem/XNW4107 against imipenem-nonsusceptible A. baumannii (n=106) was 8 mg/L, which was 16-fold lower than the MIC90 of imipenem; the resistance rate decreased from 90% to 20% applying the CLSI imipenem breakpoint. MIC90 of imipenem/XNW4107 against imipenem-resistant Klebsiella pneumoniae (n=54) was 2 mg/L, which was 128-fold lower than the MIC90 of imipenem; 80% imipenem-nonsusceptible Pseudomonas aeruginosa (n=101) exhibited MICs of imipenem/XNW4107 from 2 to 8 mg/L, which were 4- to 8-fold lower than the MICs of imipenem. Imipenem/XNW4107 was bactericidal against A. baumannii, K. pneumoniae, and Escherichia coli. The time-kill curves showed that increasing concentrations did not result in progressively increased killing at concentrations >4×MIC. Imipenem/XNW4107 has a low potential for resistance development in tested strains except for K. pneumoniae. Imipenem/XNW4107 provided good protection against imipenem-resistant A. baumannii and K. pneumoniae in vivo. The broad-spectrum profile and potent in vitro and in vivo antibacterial activities support imipenem/XNW4107 as a promising investigational candidate.

Pogostone Enhances the Antibacterial Activity of Colistin against MCR-1-Positive Bacteria by Inhibiting the Biological Function of MCR-1.

The emergence of the plasmid-mediated colistin resistance gene mcr-1 has resulted in the loss of available treatments for certain severe infections. Here we identified a potential inhibitor of MCR-1 for the treatment of infections caused by MCR-1-positive drug-resistant bacteria, especially MCR-1-positive carbapenem-resistant Enterobacteriaceae (CRE). A checkerboard minimum inhibitory concentration (MIC) test, a killing curve test, a growth curve test, bacterial live/dead assays, scanning electron microscope (SEM) analysis, cytotoxicity tests, molecular dynamics simulation analysis, and animal studies were used to confirm the in vivo/in vitro synergistic effects of pogostone and colistin. The results showed that pogostone could restore the bactericidal activity of colistin against all tested MCR-1-positive bacterial strains or MCR-1 mutant–positive bacterial strains (FIC < 0.5). Pogostone does not inhibit the expression of MCR-1. Rather, it inhibits the binding of MCR-1 to substrates by binding to amino acids in the active region of MCR-1, thus inhibiting the biological activity of MCR-1 and its mutants (such as MCR-3). An in vivo mouse systemic infection model, pogostone in combination with colistin resulted in 80.0% (the survival rates after monotherapy with colistin or pogostone alone were 33.3% and 40.0%) survival at 72 h after infection of MCR-1-positve Escherichia coli (E. coli) ZJ487 (blaNDM-1-carrying), and pogostone in combination with colistin led to one or more order of magnitude decreases in the bacterial burdens in the liver, spleen and kidney compared with pogostone or colistin alone. Our results confirm that pogostone is a potential inhibitor of MCR-1 for use in combination with polymyxin for the treatment of severe infections caused by MCR-1-positive Enterobacteriaceae.

Methamphetamine Enhances Cryptococcus neoformans Melanization, Antifungal Resistance, and Pathogenesis in a Murine Model of Drug Administration and Systemic Infection.

Methamphetamine (METH) is a major public health and safety problem in the United States. Chronic METH abuse is associated with a 2-fold-higher risk of HIV infection and, possibly, additional infections, particularly those that enter through the respiratory tract or skin. Cryptococcus neoformans is an encapsulated opportunistic yeast-like fungus that is a relatively frequent cause of meningoencephalitis in immunocompromised patients, especially in individuals with AIDS. C. neoformans melanizes during mammalian infection in a process that presumably uses host-supplied compounds such as catecholamines. l-3,4-Dihydroxyphenylalanine (l-Dopa) is a natural catecholamine that is frequently used to induce melanization in C. neoformans. l-Dopa-melanized cryptococci manifest resistance to radiation, phagocytosis, detergents, and heavy metals. Using a systemic mouse model of infection and in vitro assays to critically assess the impact of METH on C. neoformans melanization and pathogenesis, we demonstrated that METH-treated mice infected with melanized yeast cells showed increased fungal burdens in the blood and brain, exacerbating mortality. Interestingly, analyses of cultures of METH-exposed cryptococci supplemented with l-Dopa revealed that METH accelerates fungal melanization, an event of adaptation to external stimuli that can be advantageous to the fungus during pathogenesis. Our findings provide novel evidence of the impact of METH abuse on host homeostasis and increased permissiveness to opportunistic microorganisms.

A novel inhibitor of monooxygenase reversed the activity of tetracyclines against tet(X3)/tet(X4)-positive bacteria.

SummaryBackgroundTigecycline is one of the few last-resort antibiotics for the treatment of carbapenem-resistant Enterobacteriaceae infection, the incidence of which has been rapidly increasing. However, the emergence and spread of tigecycline resistance genes tet(X) (including tet(X3) and tet(X4)) has largely compromised the efficient usage of tetracyclines in the clinical settings.MethodsThe synergistic effect was determined by a checkerboard minimum inhibitory concentration (MIC) assay, a time-killing assay and scanning electron microscopy (SEM) analysis. In-depth mechanisms were defined using an enzyme inhibition assay, western blotting, RT-PCR analysis, molecular dynamics (MD) simulations, biolayer interferometry (BLI) assay and metabolomics analysis.FindingsHerein, our work identified a natural compound, plumbagin, as an effective broad-spectrum inhibitor of Tet(X) (also known as monooxygenase) by simultaneously inhibiting the activity and the production of Tet(X3)/Tet(X4). Plumbagin in combination with tetracyclines showed a synergistic bactericidal effect against Tet(X3)/Tet(X4)-producing bacteria. Mechanistic studies revealed that direct engagement of plumbagin with the catalytic pocket of Tet(X3)/Tet(X4) induced an alternation in its secondary structure to inhibit the activity of these monooxygenases. As a consequence, monotherapy or combination therapy with plumbagin increases the oxidative stress and metabolism in bacteria. Moreover, in a mouse systemic infection model of tet(X4)-positive E. coli, the combination of plumbagin and methacycline exhibited remarkable treatment benefits, as shown by a reduced bacterial load and the alleviation of pathological injury.InterpretationPlumbagin, as an inhibitor of Tet(X3)/Tet(X4), represents a promising lead drug, as well as an adjunct with tetracyclines to treat bacterial infections, especially for extensively drug-resistant bacteria harbouring Tet(X3)/Tet(X4).FundingThe National Natural Science Foundation of China.

Role of F-box Protein Cdc4 in Fungal Virulence and Sexual Reproduction of Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic yeast-like pathogen that mainly infects immunocompromised individuals and causes fatal meningitis. Sexual reproduction can promote the exchange of genetic material between different strains of C. neoformans, which is one of the reasons leading to the emergence of highly pathogenic and drug-resistant strains of C. neoformans. Although much research has been done on the regulation mechanism of Cryptococcus sexual reproduction, there are few studies on the sexual reproduction regulation of Cryptococcus by the ubiquitin-proteasome system. This study identified an F-box protein, Cdc4, which contains a putative F-box domain and eight WD40 domains. The expression pattern analysis showed that the CDC4 gene was expressed in various developmental stages of C. neoformans, and the Cdc4 protein was localized in the nucleus of cryptococcal cells. In vitro stress responses assays showed that the CDC4 overexpression strains are sensitive to SDS and MMS but not Congo red, implying that Cdc4 may regulate the cell membrane integrity and repair of DNA damage of C. neoformans. Fungal virulence assay showed that although the cdc4Δ mutant grows normally and can produce typical virulence factors such as capsule and melanin, the cdc4Δ mutant completely loses its pathogenicity in a mouse systemic-infection model. Fungal mating assays showed that Cdc4 is also essential for fungal sexual reproduction in C. neoformans. Although normal mating hyphae were observed during mating, the basidiospores’ production was blocked in bilateral mating between cdc4Δ mutants. Fungal nuclei development assay showed that the nuclei failed to undergo meiosis after fusion inside the basidia during the bilateral mating of cdc4Δ mutants, indicating that Cdc4 is critical to regulating meiosis during cryptococcal mating. In summary, our study revealed that the F-box protein Cdc4 is critical for fungal virulence and sexual reproduction in C. neoformans.

Serum apolipoprotein A-I potentiates the therapeutic efficacy of lysocin E against Staphylococcus aureus

Lysocin E is a lipopeptide with antibiotic activity against methicillin-resistant Staphylococcus aureus. For unclear reasons, the antibacterial activity of lysocin E in a mouse systemic infection model is higher than expected from in vitro results, and the in vitro activity is enhanced by addition of bovine serum. Here, we confirm that serum from various species, including humans, increases lysocin E antimicrobial activity, and identify apolipoprotein A-I (ApoA-I) as an enhancing factor. ApoA-I increases the antibacterial activity of lysocin E when added in vitro, and the antibiotic displays reduced activity in ApoA-I gene knockout mice. Binding of ApoA-I to lysocin E is enhanced by lipid II, a cell-wall synthesis precursor found in the bacterial membrane. Thus, the antimicrobial activity of lysocin E is potentiated through interactions with host serum proteins and microbial components.

The zinc cluster transcription factor Rha1 is a positive filamentation regulator in Candida albicans.

Zinc cluster transcription factors (TFs) are essential fungal regulators of gene expression. In the pathogen Candida albicans, the gene orf19.1604 encodes a zinc cluster TF regulating filament development. Hyperactivation of orf19.1604, which we have named RHA1 for Regulator of Hyphal Activity, generates wrinkled colony morphology under nonhyphal growth conditions, triggers filament formation, invasiveness, and enhanced biofilm formation and causes reduced virulence in the mouse model of systemic infection. The strain expressing activated Rha1 shows up-regulation of genes required for filamentation and cell-wall-adhesion-related proteins. Increased expression is also seen for the hyphal-inducing TFs Brg1 and Ume6, while the hyphal repressor Nrg1 is downregulated. Inactivation of RHA1 reduces filamentation under a variety of filament-inducing conditions. In contrast to the partial effect of either single mutant, the double rha1 ume6 mutant strain is highly defective in both serum- and Spider-medium-stimulated hyphal development. While the loss of Brg1 function blocks serum-stimulated hyphal development, this block can be significantly bypassed by Rha1 hyperactivity, and the combination of Rha1 hyperactivity and serum addition can generate significant polarization even in brg1 ume6 double mutants. Thus, in response to external signals, Rha1 functions with other morphogenesis regulators including Brg1 and Ume6, to mediate filamentation.

Antifungal Activity and Potential Mechanism of 6,7, 4'-O-Triacetylscutellarein Combined With Fluconazole Against Drug-Resistant C. albicans.

The increased resistance of Candida albicans to conventional antifungal drugs poses a huge challenge to the clinical treatment of this infection. In recent years, combination therapy, a potential treatment method to overcome C. albicans resistance, has gained traction. This study assessed the effect of 6,7,4′-O-triacetylscutellarein (TA) combined with fluconazole (FLC) on C. albicans in vitro and in vivo. TA combined with FLC showed good synergistic antifungal activity against drug-resistant C. albicans in vitro, with a partial inhibitory concentration index (FICI) of 0.0188–0.1800. In addition, the time-kill curve confirmed the synergistic effect of TA and FLC. TA combined with FLC showed a strong synergistic inhibitory effect on the biofilm formation of resistant C. albicans. The combined antifungal efficacy of TA and FLC was evaluated in vivo in a mouse systemic fungal infection model. TA combined with FLC prolonged the survival rate of mice infected with drug-resistant C. albicans and reduced tissue invasion. TA combined with FLC also significantly inhibited the yeast-hypha conversion of C. albicans and significantly reduced the expression of RAS-cAMP-PKA signaling pathway-related genes (RAS1 and EFG1) and hyphal-related genes (HWP1 and ECE1). Furthermore, the mycelium growth on TA combined with the FLC group recovered after adding exogenous db-cAMP. Collectively, these results show that TA combined with FLC inhibits the formation of hyphae and biofilms through the RAS-cAMP-PKA signaling pathway, resulting in reduced infectivity and resistance of C. albicans. Therefore, this study provides a basis for the treatment of drug-resistant C. albicans infections.