Murine Infection Model Research Articles

Irradiated whole cell Chlamydia vaccine confers significant protection in a murine genital tract challenge model

Abstract Chlamydia trachomatis infections are the most common bacterial STIs globally and can lead to serious morbidity if untreated. Development of a killed, whole-cell vaccine has been stymied by coincident epitope destruction during inactivation. Here, we present a prototype Chlamydia vaccine composed of elementary bodies (EBs) from the related mouse pathogen, Chlamydia muridarum (Cm). EBs inactivated by gamma rays (Ir-Cm) in the presence of the antioxidant Mn2+-Decapeptide (DEHGTAVMLK) Phosphate (MDP) are protected from epitope damage but not DNA damage. Cm EBs gamma-inactivated with MDP retain their structure and provide significant protection in a murine genital tract infection model. Mice vaccinated with Ir-Cm (+MDP) exhibited elevated levels of Cm-specific IgG and IgA antibodies, reduced bacterial burdens, accelerated clearance, and distinctive cytokine responses compared to unvaccinated controls and animals vaccinated with EBs irradiated without MDP. Preserving EB epitopes with MDP during gamma inactivation offers the potential for a polyvalent, whole-cell vaccine against C. trachomatis.

Cell aggregation mediated by ACE2 deletion in Candida auris modulates fungal colonization and host immune responses in the skin.

Candida auris is an emerging multi-drug-resistant fungal pathogen that colonizes the skin and causes invasive infections in hospitalized patients. Multi-cellular aggregative phenotype is widely reported in the C. auris isolates, but its role in skin colonization and host immune response is not yet known. In this study, we generated aggregative phenotype by deleting the ACE2 gene in C. auris and determined the fungal colonization and host immune response using an intradermal mouse model of C. auris skin infection. Our results indicate that mice infected with ace2Δ strain had significantly lower fungal load after 3 and 14 days post-infections compared to the non-aggregative wild-type and the ACE2 reintegrated strain. The colonization of ace2Δ is associated with increased recruitment of CD11b+ Ly6G+ neutrophils and decreased accumulation of CD11b+ Ly6 Chi inflammatory monocytes and CD11b+ MHCII+ CD64+ macrophages. Furthermore, Th17 cells and type 3 innate lymphoid cells (ILCs) were significantly increased in the skin tissue of ace2Δ infected mice. Our findings suggest that aggregative phenotype mediated by ACE2 deletion in C. auris induces potent neutrophil and IL-17-mediated immune response and reduces fungal colonization in the skin.IMPORTANCEC. auris is a rapidly emerging fungal pathogen that can colonize hospitalized patients, especially in skin tissue, and cause invasive infections. C. auris isolates exhibit morphological heterogeneity, and the multicellular aggregative phenotype of C. auris is reported frequently in clinical settings. Understanding the role of fungal morphotypes in colonization, persistence, and immune response in the skin microenvironment will have potential applications in clinical diagnosis and novel preventive and therapeutic measures. Here, we utilized the murine model of intradermal infection and determined that the aggregative phenotype of C. auris as the result of ACE2 gene deletion elicits potential innate and adaptive immune responses in mice. These observations will help explain the differences in the skin colonization and immune responses of the aggregative morphotype of C. auris and open the door to developing novel antifungal therapeutics.

Establishment of a diverse pheno-genotypic challenge set of Klebsiella pneumoniae and Pseudomonas aeruginosa suitable for use in the murine pneumonia model.

Preclinical murine infection models lack inter-laboratory uniformity, complicating result comparisons and data reproducibility. The European Innovative Medicines initiative-funded consortium (COMBINE) has developed a standardized murine neutropenic pneumonia protocol to address these concerns. While model methods have been standardized, a major obstacle to consistent results is the lack of available bacteria with defined viability and variability. Herein, we establish a diverse challenge set of Klebsiella pneumoniae and Pseudomonas aeruginosa suitable for use in the COMBINE protocol to further minimize experimental inconsistency and improve the interpretability of data generated among differing laboratories. Sixty-six K. pneumoniae and 65 P. aeruginosa were phenotypically profiled against tigecycline (K. pneumoniae only), levofloxacin, meropenem, cefiderocol and tobramycin. Fifty-nine isolates were introduced into the COMBINE model to assess the sufficiency of the starting bacterial inoculation, resultant baseline bacterial burden, achievement of ≥1 log10cfu/lung growth at 24 h, time to and percentage mortality. Forty-five isolates displaying desirable minimum inhibitory concentration profiles were subjected to replicate in vivo testing to assess target parameters. 83% of K. pneumoniae reached the prerequisite growth at 24 h using a starting bacterial burden ≥7 log10cfu/lung. P. aeruginosa isolates grew well in the model: 90% achieved the growth target with a starting bacterial burden of 6 log10cfu/lung. Mortality was negligible for K. pneumoniae but high for P. aeruginosa. Poor or inconsistent achievement of the 24 h growth target was seen in 11/59 isolates. With this diverse cache of viable isolates established in the COMBINE pneumonia model, future translational studies can be undertaken to set efficacy benchmarks among laboratories.

The Antifungal Effects of Berberine and Its Proposed Mechanism of Action Through CYP51 Inhibition, as Predicted by Molecular Docking and Binding Analysis

Fungal infections present a significant health risk, particularly in immunocompromised individuals. Berberine, a natural isoquinoline alkaloid, has demonstrated broad-spectrum antimicrobial activity, though its antifungal potential and underlying mechanisms against both yeast-like and filamentous fungi are not fully understood. This study investigates the antifungal efficacy of berberine against Candida albicans, Cryptococcus neoformans, Trichophyton rubrum, and Trichophyton mentagrophytes in vitro, as well as its therapeutic potential in a murine model of cryptococcal infection. Berberine showed strong antifungal activity, with MIC values ranging from 64 to 128 µg/mL. SEM and TEM analyses revealed that berberine induced notable disruptions to the cell wall and membrane in C. neoformans. No signs of cell necrosis or apoptosis were observed in fungal cells treated with 2 × MIC berberine, and it did not increase intracellular ROS levels or affect mitochondrial membrane potential. Molecular docking and binding affinity assays demonstrated a strong interaction between berberine and the fungal enzyme CYP51, with a dissociation constant (KD) of less than 1 × 10−12 M, suggesting potent inhibition of ergosterol biosynthesis. In vivo studies further showed that berberine promoted healing in guinea pigs infected with T. mentagrophytes, and in a murine cryptococcal infection model, it prolonged survival and reduced lung inflammation, showing comparable efficacy to fluconazole. These findings indicate that berberine exerts broad-spectrum antifungal effects through membrane disruption and CYP51 inhibition, highlighting its potential as a promising therapeutic option for fungal infections.

Synergistic wall digestion and cuproptosis against fungal infections using lywallzyme-induced self-assembly of metal-phenolic nanoflowers

Fungi are very common infectious pathogens, which may cause invasive and potentially life-threatening infections. However, the efficacy of antifungal medications remains limited. Herein, a Cu2+-phenolic nanoflower is designed to combat fungal infections by combining cuproptosis and cell wall digestion. Firstly, protocatechuic acid (PA)-Cu2+ (PC) nanopetals are prepared by coordination interaction. Lywallzyme (Lyw) is then added to induce the self-assembly of PC to form Lyw loaded PC (PCW) nanoflowers. PCW nanoflowers can effectively adhere to fungal surface and Lyw can digest fungal cell walls to facilitate Cu2+ to penetrate into fungal interior, thereby exerting a synergistic fungicidal effect. PCW nanoflowers exhibit excellent fungicidal activity even in protein-rich and high-salt conditions, where dissociative Cu2+ completely loses fungicidal activity. Transcriptome sequencing analysis reveals that PCW can lead to fungal cuproptosis. The in vivo fungicidal effect of PCW nanoflowers is confirmed on a murine skin fungal infection model and a murine fungal keratitis model.

Assays for Assessing Mycobacterium avium Immunity and Evaluating the Effects of Therapeutics.

In Europe and North America, the prevalence of pulmonary nontuberculous mycobacteria (NTM) is increasing. Most pulmonary NTM infections are caused by the Mycobacterium avium complex (MAC). Sadly, the treatment of pulmonary MAC is suboptimal with failure rates ranging from 37% to 58%. Therefore, there is a need to develop new therapeutics. Developing new immunotherapies and studying their interaction with standard or new drugs requires reliable assays. Four different assays including CFSE-based flow cytometry, in vitro protection assays, IFN-γ ELISPOT, and murine infection models were optimized using a reference strain of MAC (ATCC 700898) to help with the development of immunotherapies for MAC. Expansion of proliferating and IFN-γ producing human T cells is optimal after 7 days of stimulation with MAC at a multiplicity of infection (MOI) of 0.1, achieving a stimulation index of 26.5 ± 11.6 (mean ± SE). The in vitro protection assay for MAC works best by co-culturing T cells expanded for 7 days with MAC (MOI 1)-infected autologous macrophages. Aerosol MAC infection of mice allows measurement of the effects of the BCG vaccine and clarithromycin. IFN-γ ELISPOT assays with live MAC (MOI 3) stimulation of splenocytes from mice immunized with BCG help identify differences between unimmunized mice and mice immunized with BCG. In conclusion, multiple assays are available for use to identify MAC-specific effector T cells, which will help in the development of new therapeutics or vaccines against pulmonary MAC.

Lung Damage Induced by Plasmodium berghei ANKA in Murine Model of Malarial Infection is Mitigated by Dietary Supplementation with DHA-Rich Omega-3.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are severe complications that can occur in infections caused by any Plasmodium species. Due to the high lethality rate and the lack of specific treatment for ALI/ARDS, studies aimed at understanding and searching for treatment strategies for such complications have been fundamental. Here, we investigated the protective role of dietary supplementation with DHA-rich fish oil against lung damage induced by Plasmodium berghei ANKA in a murine model. Our results demonstrated that alveolar vascular damage, lung edema, and histopathological alterations were significantly reduced in mice that received dietary supplementation compared to those that did not receive the supplementation. Furthermore, a significant reduction in the number of CD8+ T lymphocytes, in addition to reduced infiltration of inflammatory cells in the bronchoalveolar lavage fluid was also observed. High levels of IL-10, but not of TNF-α and IFN-γ, were also observed in infected mice that received the supplementation, along with a reduction in local oxidative stress. Together, the data suggest that dietary supplementation with DHA-rich fish oil in malarial endemic areas may help reduce lung damage resulting from the infection, thus preventing worsening of the condition.

Can letrozole be repurposed for the treatment of visceral leishmaniasis?

Visceral leishmaniasis, caused by Leishmania infantum in New World countries, is the most serious and potentially fatal form of leishmaniasis, if left untreated. There are currently no effective prophylactic measures, and therapeutic options are limited. Therefore, we investigated whether the aromatase inhibitor letrozole (LET), which is already used to treat breast cancer, has an antileishmanial activity and/or immunomodulatory potential and therefore may be used to treat L. infantum infection. LET was active against L. infantum promastigote and amastigote life cycle stages in an in vitro infection model using human THP-1 cell-derived macrophages. In human peripheral blood leukocytes ex vivo, LET reduced the internalized forms of L. infantum by classical monocytes and activated neutrophils. Concomitantly, LET stimulated the production of IL-12/TNF-α and decreased the production of IL-10/TGF-β by peripheral blood phagocytes, while in T and B cells, it promoted the production of TNF-α/IFN-γ and decreased that of IL-10. In a murine infection model, LET significantly reduced the parasite load in the liver after just 5 days and in the spleen after 15 days. During in vivo treatment with LET, the production of TNF-α/IFN-γ also increased. In addition, the proportion of developing granulomas decreased and that of mature granulomas increased in the liver, while there was no significant change in organ architecture in the spleen. Based on these data, repositioning of LET may be promising for the treatment of visceral leishmaniasis in humans.

Zidovudine in synergistic combination with nitrofurantoin or omadacycline: in vitro and in murine urinary tract or lung infection evaluation against multidrug-resistant Klebsiella pneumoniae.

Limited treatment options and multidrug-resistant (MDR) Klebsiella pneumoniae present a significant therapeutic challenge, underscoring the need for novel approaches. Drug repurposing is a promising tool for augmenting the activity of many antibiotics. This study aimed to identify novel synergistic drug combinations against K. pneumoniae based on drug repurposing. We used the clinically isolated GN 172867 MDR strain of K. pneumoniae to determine the reversal resistance activity of zidovudine (AZT). The combined effects of AZT and various antibiotics, including nitrofurantoin (NIT) and omadacycline (OMC), were examined using the checkerboard method, growth curves, and crystal violet assays to assess biofilms. An in vitro combination activity testing was carried out in 12 isolates of K. pneumoniae. In vivo murine urinary tract and lung infection models were used to evaluate the therapeutic effects of AZT + NIT and AZT + OMC, respectively. The fractional inhibitory concentration index and growth curve demonstrated that AZT synergized with NIT or OMC against K. pneumoniae strains. In addition, AZT + NIT inhibited biofilm formation and cleared mature biofilms. In vivo, compared with untreated GN 172867-infected mice, AZT + NIT and AZT + OMC treatment decreased colony counts in multiple tissues (P < 0.05) and pathological scores in the bladder and kidneys (P < 0.05) and increased the survival rate by 60% (P < 0.05). This study evaluated the combination of AZT and antibiotics to treat drug-resistant K. pneumoniae infections and found novel drug combinations for the treatment of acute urinary tract infections. These findings suggest that AZT may exert significant anti-resistance activity.

Fatty acid metabolism in neutrophils promotes lung damage and bacterial replication during tuberculosis.

Mycobacterium tuberculosis (Mtb) infection induces a marked influx of neutrophils into the lungs, which intensifies the severity of tuberculosis (TB). The metabolic state of neutrophils significantly influences their functional response during inflammation and interaction with bacterial pathogens. However, the effect of Mtb infection on neutrophil metabolism and its consequent role in TB pathogenesis remain unclear. In this study, we examined the contribution of glycolysis and fatty acid metabolism on neutrophil responses to Mtb HN878 infection using ex-vivo assays and murine infection models. We discover that blocking glycolysis aggravates TB pathology, whereas inhibiting fatty acid oxidation (FAO) yields protective outcomes, including reduced weight loss, immunopathology, and bacterial burden in lung. Intriguingly, FAO inhibition preferentially disrupts the recruitment of a pathogen-permissive immature neutrophil population (Ly6Glo/dim), known to accumulate during TB. Targeting carnitine palmitoyl transferase 1a (Cpt1a)-a crucial enzyme in mitochondrial β-oxidation-either through chemical or genetic methods impairs neutrophils' ability to migrate to infection sites while also enhancing their antimicrobial function. Our findings illuminate the critical influence of neutrophil immunometabolism in TB pathogenesis, suggesting that manipulating fatty acid metabolism presents a novel avenue for host-directed TB therapies by modulating neutrophil functions.

The Endocytosis Adaptor Sla1 Facilitates Drug Susceptibility and Fungal Pathogenesis Through Sla1-Efg1 Regulating System in Candida albicans.

The role of endocytosis in Candida albicans drug-resistance and pathogenicity remains poorly understood, despite its importance as a fundamental component of intracellular trafficking. In order to understand the role of endocytosis in Candida albicans cell wall integrity, drug resistance, and virulence. Detection of intracellular endocytosis by FM4-64 staining; Scanning electron microscopy is used to detect cell wall components; Spot assay for detecting drug sensitivity; Co-ip is used to detect protein interactions. In this study, we found the functions of Sla1 in regulating endocytosis is conserved among pathogenic fungi. Our results also revealed that the deletion of the SLA1 gene altered cell wall properties, composition, and gene expression. In addition, we showed that C. albicans Sla1 was responsible for hyphal development in vitro and for fungal pathogenicity in a murine infection model. Intriguingly, sla1∆/∆ mutant demonstrated enhanced drug resistance, and Sla1 was found to interact with the transcription factor Efg1; the relationship between Sla1 and Efg1 impacts the expression of genes encoding components of the ergosterol biosynthesis pathway, including ERG1, EGR11, and ERG25. These findings have expanded our knowledge of the capabilities of Sla1 beyond its role as an endocytosis adapter and provided insights into a potential new therapeutic target for the treatment of fungal infections.

‘GGFGGQ’ repeats in Hfq of Acinetobacter baumannii are essential for nutrient utilization and virulence

The nosocomial pathogen Acinetobacter baumannii is known for causing lung and soft tissue infections in immunocompromised hosts. Its ability to adapt to various environments through post-transcriptional gene regulation is key to its success. Central to this regulation is the RNA chaperone Hfq, which facilitates interactions between mRNA targets and their small RNA (sRNA) partners through a Sm-core domain. Notably, the A. baumannii Hfq protein has a uniquely long C-terminal domain (CTD) with GGFGGQ amino acid repeats and an acidic amino acid-rich C-terminal tip (C-tip). Previous research has shown the importance of the intact CTD for Hfq's functionality. Given the significance of the C-tip in E. coli Hfq, we examined the pathophysiological roles of the redundant 'GGFGGQ' repeats along with the C-tip of A. baumannii Hfq. We constructed several variations of Hfq protein with fewer 'GGFGGQ' repeats while preserving the C-tip, and variants with altered C-tip amino acid composition. We then studied their RNA interaction abilities and assessed the pathophysiological fitness and virulence of genome-complemented A. baumannii mutants. Our findings reveal that the redundancy of the 'GGFGGQ' repeats is crucial for Hfq's role in pathophysiological fitness and negatively impacts A. baumannii's virulence in a murine lung infection model. In addition, C-tip mutants exhibited a negative effect on both fitness and virulence, however, to a lesser extent than the other variants. These results underscore the importance of 'GGFGGQ' redundancy and acidic residues in Hfq's ribo-regulation and autoregulation, suggesting their critical role in establishing regulatory networks.

Vaginal Tritrichomonas foetus infection in mice as an in vivo model for drug development against Trichomonas vaginalis.

Trichomonas vaginalis is the causative agent of the common sexually transmitted disease, trichomoniasis, which affects more than a hundred million people worldwide. Metronidazole and tinidazole, agents belonging to the 5-nitroheterocyclic class of antimicrobials, are most often used to treat infection, but increased resistance has been reported and adverse effects of these drugs can be significant. Consequently, an urgent need exists for the development of novel drug entities against trichomoniasis. Critical for antimicrobial drug development is the demonstration of in vivo efficacy. Murine models of vaginal T. vaginalis infection are unreliable for unknown reasons. Meanwhile, murine infections with the related bovine pathogen, Tritrichomonas foetus, tend to be more robust, although susceptibility to different antimicrobials might differ from T. vaginalis. Here, we explored the utility of T. foetus infection as a surrogate model for drug development against T. vaginalis. Four different T. foetus strains caused robust vaginal infection in young mice, while none of four diverse T. vaginalis strains did. Comparison of drug susceptibility profiles revealed that T. foetus and T. vaginalis were similarly susceptible to a range of 5-nitroheterocyclic and gold(I) compounds. By comparison, proteasome inhibitors were 10- to 15-fold less active against T. foetus than T. vaginalis, although one of the proteasome inhibitors, bortezomib, had low micromolar activity or better against multiple strains of both trichomonads. Different strains of T. foetus were used to demonstrate the utility of the murine vaginal infection models for in vivo efficacy testing, including for bortezomib and a gold(I) compound. The differences in susceptibility to proteasome inhibitors may be partially explained by differences in the proteasome subunit sequences between the two trichomonads, although the functional relevance of the proteasome was similar in both organisms. These findings indicate that T. foetus can serve as a reliable surrogate model for T. vaginalis in vitro and in murine infections in vivo, but caution must be exercised for specific drug classes with targets, such as the proteasome, that may display genetic divergence between the trichomonads.

Electrically-driven drug delivery into deep cutaneous tissue by conductive microneedles for fungal infection eradication and protective immunity

Fungal infections affect over 13 million people worldwide and are responsible for 1.5 million deaths annually. Some deep cutaneous fungal infections may extend the dermal barriers to cause systemic infection, resulting in substantial morbidity and mortality. However, the management of deep cutaneous fungal infection is challenging and yet overlooked by traditional treatments, which only offer limited drug availability within deep tissue. In this study, we have developed an electrically stimulated microneedle patch to deliver miconazole into the subcutaneous layer. We tested its antifungal efficacy using in vitro and ex vivo models that mimic fungal infection. Moreover, we confirmed its anti-fungal and wound-healing effects in a murine subcutaneous fungal infection model. Furthermore, our findings also showed that the combination of miconazole and applied current synergistically stimulated the nociceptive sensory nerves, thereby activating protective cutaneous immunity mediated by dermal dendritic and γδ-T cells. Collectively, this study provides a new strategy for minimally invasive delivery of therapeutic agents and the modulation of the neuro-immune axis in deep tissue.

Antibacterial and mechanism of action studies of boxazomycin A.

Boxazomycins A-C are potent broad-spectrum antibiotics isolated from Actinomycetes strain G495-1 in 1987. We now report that boxazomycin A inhibits bacterial growth by selectively inhibiting protein synthesis, its effect is bacteriostatic, and it is equally active against drug resistant bacterial strains. No cross-resistance to protein synthesis inhibitors was observed suggesting that its inhibition is distinct from clinical protein synthesis inhibitors. We also report in vivo efficacy in a Staphylococcus aureus murine infection model supported by corresponding pharmacokinetic studies.

Efficacy of mefloquine and its enantiomers in a murine model of Mycobacterium avium infection.

The treatment of Mycobacterium avium infections is still long, complex, and often poorly tolerated, besides emergence of resistances. New active molecules that are more effective and better tolerated are deeply needed. Mefloquine and its enantiomers ((+) Erythro-mefloquine ((+)-EMQ) and (-)-Erythro-mefloquine ((-)-EMQ)) have shown efficacy in both in vitro and in vivo, in a mouse model of M. avium intraveinous infection. However, no study reports aerosol model of infection or combination with gold standard treatment. That was the aim of our study. In an aerosol model of M. avium infection in BALB/c mice, we used five treatment groups as followed: Clarithromycin-Ethambutol-Rifampicin (CLR-EMB-RIF, standard of care, n = 15), CLR-EMB-MFQ (n = 15), CLR-EMB-(+)-EMQ (n = 15), CLR-EMB-(-)-EMQ (n = 15) and an untreated group (n = 25). To evaluate drug efficacy, we sacrificed each month over 3 months, 5 mice from each group. Lung homogenates were diluted and plated for colony forming unit count (CFU) expressed in Log10. At each time point, we found a significant difference between the untreated group and each of the treatment groups (p<0.005). The (+)-EMQ-CLR-EMB group was the group with the lowest CFU count at each time point but never reached statistical significance. The results of each group 3 months after treatment are: (+)-EMQ-CLR-EMB (4.43 ± 0.26), RIF-CLR-EMB (4.83 ± 0.37), (-)-EMQ-CLR-EMB (4.82 ± 0.18), MFQ-CLR-EMB (4.70 ± 0.21). In conclusion, MFQ and its enantiomers appear to be as effective as rifampicin in combination therapy. Further studies are needed to evaluate the ability of these drugs to prevent selection of clarithromycin resistant strains and potential for lung sterilization.

Spectinamide MBX-4888A exhibits favorable lesion and tissue distribution and promotes treatment shortening in advanced murine models of tuberculosis.

The spectinamides are novel, narrow-spectrum semisynthetic analogs of spectinomycin, modified to avoid intrinsic efflux by Mycobacterium tuberculosis. Spectinamides, including lead MBX-4888A (Lee-1810), exhibit promising therapeutic profiles in mice, as single drugs and as partner agents with other anti-tuberculosis antibiotics including rifampin and/or pyrazinamide. Here, we show that MBX-4888A, given by injection with the front-line standard of care regimen, is treatment shortening in multiple murine tuberculosis infection models. The positive treatment responses to MBX-4888A combination therapy in multiple mouse models, including mice exhibiting advanced pulmonary disease, can be attributed to favorable distribution in tissues and lesions, retention in caseum, along with favorable effects with rifampin and pyrazinamide under conditions achieved in necrotic lesions. This study also provides an additional data point regarding the safety and tolerability of spectinamide MBX-4888A in long-term murine efficacy studies.

Pneumolysin contributes to dysfunction of nasal epithelial barrier for promotion of pneumococcal dissemination into brain tissue.

Streptococcus pneumoniae is one of the major pathogens responsible for bacterial meningitis and neurological sequelae. The present study was conducted to identify a non-hematogenous route used by S. pneumoniae to gain access to brain tissue without causing bacteremia or pneumonia, as well as bacterial and host factors involved in this process. To investigate the molecular mechanisms and dissemination pathways of pneumococcal infection in brain tissue, mice were intranasally inoculated with S. pneumoniae strain EF3030, a clinical isolate from a patient with otitis media. Pneumococci were isolated from the frontal olfactory bulb, caudal cerebrum, and cerebellum, with neither bacteremia nor pneumonia observed in the present model. Immunostaining imaging revealed the presence of S. pneumoniae organisms in olfactory nerve fibers. Knockout of the ply gene encoding pneumolysin (PLY) markedly compromised the ability of the bacterial organisms to disseminate into brain tissue, whereas the dissemination efficiency of the complemented strain was restored to nearly the same level as the wild type. Notably, distinct upregulation of Gli1 and Snail1, which are involved in the transcriptional repression of junctional proteins, along with downregulation of E-cadherin, was detected in nasal lavage samples from mice infected with the wild-type or complemented strain, but not in those from mice infected with the ply mutant. Taken together, the present findings indicate that PLY induces Gli1-Snail1-dependent dysfunction of the nasal epithelial barrier, thus allowing pneumococcal dissemination to brain tissue that occurs in a non-hematogenous manner.IMPORTANCEBacterial meningitis, considered to be caused by bacteremia, can lead to blood-brain barrier disruption and bacterial dissemination into the central nervous system. Despite the availability of intravenously administered antibiotics with cerebrospinal fluid transferability, bacterial meningitis remains associated with high rates of morbidity and mortality. Here, we utilized Streptococcus pneumoniae strain EF3030, clinically isolated from otitis media, for the construction of a murine infection model to investigate the molecular mechanisms by which nasally colonized pneumococci disseminate into brain tissue. The obtained findings indicate that pneumolysin (PLY) induces Gli1-Snail1-dependent dysfunction of the nasal epithelial barrier, which facilitates pneumococcal dissemination to brain tissue in a non-hematogenous manner. Our results support the existence of an alternative route by which S. pneumoniae can reach the central nervous system and indicate the need for the development of novel therapeutic strategies, which would be an important contribution to the clinical management of bacterial meningitis.

Altered oral microbiota of drug-resistant organism carriers exhibit impaired gram-negative pathogen inhibition.

The oral microbiome has been understudied as a reservoir for clinical pathogens, including drug-resistant strains. Understanding how alterations in microbiome functioning render this site vulnerable to colonization is essential, as multidrug-resistant organisms (MDRO) carriage is a major risk factor for developing serious infections. To advance our knowledge of oral MDRO carriage and protection against pathogen colonization conferred by native microbiota, we examined microbiomes from individuals colonized by MDROs (n=33) and non-colonized age-matched controls (n=30). Shotgun metagenomic analyses of oral swabs from study participants revealed significant differences in microbial communities with depletion of Streptococcus spp. among those colonized by multidrug-resistant gram-negative bacilli (RGNB), compared to non-carriers. We utilized metagenomic sequencing to characterize the oral resistome and find antimicrobial resistance genes are present in higher abundance among RNGB carriers versus non-carriers. High-throughput co-culture screening revealed oral bacteria isolated from MDRO non-carriers demonstrate greater inhibition of gram-negative pathogens, compared to isolates from carriers. Moreover, biosynthetic gene clusters from streptococci are found in higher abundance from non-carrier microbiomes, compared to RGNB carrier microbiomes. Bioactivity-guided fractionation of extracts from Streptococcus isolate SID2657 demonstrated evidence of strong E. coli and A. baumannii inhibition in a murine model of infection. Together, this provides evidence that oral microbiota shape this dynamic microbial community and may serve as an untapped source for much-needed antimicrobial small-molecules.

Disruption to de novo uridine biosynthesis alters β-1,3-glucan masking in Candida albicans.

The uridine derivatives UDP-glucose and UDP-N-acetylglucosamine are important for cell wall construction as they are the precursors for the synthesis of β-1,3-glucan and chitin, respectively. Previous studies have demonstrated attenuated virulence of uridine auxotrophs in mice, which has been attributed to insufficient uridine levels for growth in the host. We have discovered that uridine deprivation in the uridine auxotroph ura3ΔΔ disrupts cell wall architecture by increasing surface mannans, exposing β-1,3-glucan and chitin, and decreasing UDP-sugar levels. Cell wall architecture and UDP-sugars can be rescued with uridine supplementation. The cell wall architectural disruptions in the ura3ΔΔ mutant also impact immune activation since the mutant elicited greater TNFα secretion from RAW264.7 macrophages than wild type. To determine if cell wall defects contributed to decreased virulence in the ura3ΔΔ mutant, we used a murine model of systemic infection. Mice infected with the ura3ΔΔ mutant exhibited increased survival and reduced kidney fungal burden compared with mice infected with wild type. However, suppression of the immune response with cyclophosphamide did not rescue virulence in mice infected with the ura3ΔΔ mutant, indicating the attenuation in virulence of uridine auxotrophs can be attributed to decreased growth in the host but not increased exposure of β-1,3-glucan. Moreover, the ura3ΔΔ mutant is unable to grow on ex vivo kidney agar, which demonstrates its inability to colonize the kidneys due to poor growth. Thus, although uridine auxotrophy elicits changes to cell wall architecture that increase the exposure of immunogenic polymers, metabolic fitness costs more strongly drive the observed virulence attenuation.IMPORTANCECandida albicans is a common cause of bloodstream infections (candidemia). Treatment of these bloodstream infections is made difficult because of increasing antifungal resistance and drug toxicity. Thus, new tactics are needed for antifungal drug development, with immunotherapy being of particular interest. The cell wall of C. albicans is composed of highly immunogenic polymers, particularly β-1,3-glucan. However, β-1,3-glucan is naturally masked by an outer layer of mannoproteins, which hampers the detection of the fungus by the host immune system. Alteration in cell wall components has been shown to increase β-1,3-glucan exposure; however, it is unknown how the inability to synthesize precursors to cell wall components affects unmasking. Here, we demonstrate how cell wall architecture is altered in response to a deficit in precursors for cell wall synthesis and how uridine is a crucial component of these precursors.