A fungal metabolic regulator underlies infectious synergism during Candida albicans-Staphylococcus aureus intra-abdominal co-infection.

Candida albicans and Staphylococcus aureus are two commonly associated pathogens that cause nosocomial infections with high morbidity and mortality. Our prior and current work using a murine model of polymicrobial intra-abdominal infection (IAI) demonstrates that synergistic lethality is driven by Candida-induced upregulation of functional S. aureus α-toxin leading to polymicrobial sepsis and organ damage. In order to determine the candidal effector(s) mediating enhanced virulence, an unbiased screen of C. albicans transcription factor mutants was undertaken revealing that zcf13Δ/Δ fails to drive augmented α-toxin or lethal synergism during co-infection. A combination of transcriptional and phenotypic profiling approaches shows that ZCF13 regulates genes involved in pentose metabolism, including RBK1 and HGT7 that contribute to fungal ribose catabolism and uptake, respectively. Subsequent experiments reveal that ribose inhibits the staphylococcal agr quorum sensing system and concomitantly represses toxicity. Unlike wild-type C. albicans, zcf13Δ/Δ did not effectively utilize ribose during co-culture or co-infection leading to exogenous ribose accumulation and agr repression. Forced expression of RBK1 and HGT7 in the zcf13Δ/Δ mutant fully restores pathogenicity during co-infection. Collectively, our results detail the interwoven complexities of cross-kingdom interactions and highlight how intermicrobial metabolism impacts polymicrobial disease pathogenesis with devastating consequences for the host.

Silver Diammine Fluoride Usage in General Dentistry Offices in Louisiana.

Purpose: To evaluate the current knowledge and usage of silver diammine fluoride (SDF) by general dentists in Louisiana and to identify primary barriers to the imple- mentation of SDF. Methods: A 16-item survey was emailed to 1719 Louisiana Dental Association members to identify factors influencing general dentists' usage of SDF. Results: Eighty-two surveys were completed with a response rate of 4.8 percent, with 69 identified as general dentists. Over half of the respondents were male (53.6%) and their practice experience ranged from less than one year to 48 years. The majority were solo owners (43.5%) while 7.3 percent had jobs in the corporate setting. Most agreed/strongly agreed that their knowledge of SDF was from either dental journals or online resources, while fewer stated they were taught about SDF (25%) or used SDF (8%) in dental school. The majority knew the advantages and off-label usage of SDF. However, only 40 percent recognized that SDF was officially approved for tooth hypersensitivity only. The most reported perceived barrier to SDF implementation was not learning about SDF in dental school (36%). Conclusion: There is a lack of understanding of SDF usage among Louisiana general dentists. The main reason for not incorporating SDF into their practice is the lack of training in their dental education.

A fungal metabolic regulator underlies infectious synergism during Candida albicans - Staphylococcus aureus intra-abdominal co-infection.

Candida albicans and Staphylococcus aureus are two commonly associated pathogens that cause nosocomial infections with high morbidity and mortality. Our prior and current work using a murine model of polymicrobial intra-abdominal infection (IAI) uncovered synergistic lethality that was driven by Candida -induced upregulation of functional S. aureus ⍺-toxin leading to polymicrobial sepsis and organ damage. In order to determine the candidal effector(s) mediating enhanced virulence, an unbiased screen of C. albicans transcription factor mutants was undertaken and revealed that zcf13 Δ/Δ failed to drive augmented ⍺-toxin or lethal synergism during co-infection. Using a combination of transcriptional and phenotypic profiling approaches, ZCF13 was shown to regulate genes involved in pentose metabolism, including RBK1 and HGT7 that contribute to fungal ribose catabolism and uptake, respectively. Subsequent experiments revealed that ribose inhibited the staphylococcal agr quorum sensing system and concomitantly repressed toxicity. Unlike wild-type C. albicans , zcf13 Δ/Δ was unable to effectively utilize ribose during co-culture or co-infection leading to exogenous ribose accumulation and agr repression. Forced expression of RBK1 and HGT7 in the zcf13 Δ/Δ mutant fully restored pathogenicity during co-infection. Collectively, our results detail the interwoven complexities of cross-kingdom interactions and highlight how intermicrobial metabolism impacts polymicrobial disease pathogenesis with devastating consequences for the host.

Impaired neutrophil extracellular trap-forming capacity contributes to susceptibility to chronic vaginitis in a mouse model of vulvovaginal candidiasis.

Vulvovaginal candidiasis (VVC), caused by Candida albicans, is characterized by aberrant inflammation by polymorphonuclear neutrophils (PMNs) in the vaginal lumen. Data from the established murine model shows that despite potent antifungal properties, PMNs fail to clear C. albicans due to local heparan sulfate that inhibits the interaction between PMNs and C. albicans, resulting in chronic vaginal immunopathology. To understand the role of neutrophil extracellular traps (NETs) in defense against C. albicans at the vaginal mucosa, we investigated the NET-forming capacity of PMNs in chronic VVC-susceptible (CVVC-S/C3H) and -resistant (CVVC-R/CD-1) mouse strains. Immunofluorescence revealed the formation of NETs (release of DNA with PMN-derived antimicrobial proteins) in PMN-C. albicans cocultures using vaginal conditioned medium (VCM) generated from CVVC-R/CD-1 mice, similar to NET-inducing positive controls. Under these NETotic conditions, PMNs released high levels of double-stranded DNA bound with NET-associated proteins, concomitant with substantial C. albicans killing activity. In contrast, PMN-C. albicans cocultures in VCM from CVVC-S/C3H mice lacked NET formation together with reduced antifungal activity. Similar results were observed in vivo: active NET-C. albicans interaction followed by fungal clearance in inoculated CVVC-R/CD-1 mice, and sustained high vaginal fungal burden and no evidence of NETs in inoculated CVVC-S/C3H mice. Furthermore, the level of Ki67 expression, a putative NETotic PMN marker, was significantly reduced in vaginal lavage fluid from CVVC-S/C3H mice compared to CVVC-R/CD-1 mice. Finally, scanning electron microscopy revealed that PMNs in CVVC-R, but not CVVC-S, conditions exhibited NETs in direct contact with C. albicans hyphae in vitro and in vivo. These results suggest that VVC-associated immunopathology includes impaired NET-mediated antifungal activity.

Candida-induced granulocytic myeloid-derived suppressor cells are protective against polymicrobial sepsis.

IMPORTANCE: Polymicrobial intra-abdominal infections are serious clinical infections that can lead to life-threatening sepsis, which is difficult to treat in part due to the complex and dynamic inflammatory responses involved. Our prior studies demonstrated that immunization with low-virulence Candida species can provide strong protection against lethal polymicrobial sepsis challenge in mice. This long-lived protection was found to be mediated by trained Gr-1+ polymorphonuclear leukocytes with features resembling myeloid-derived suppressor cells (MDSCs). Here we definitively characterize these cells as MDSCs and demonstrate that their mechanism of protection involves the abrogation of lethal inflammation, in part through the action of the anti-inflammatory cytokine interleukin (IL)-10. These studies highlight the role of MDSCs and IL-10 in controlling acute lethal inflammation and give support for the utility of trained tolerogenic immune responses in the clinical treatment of sepsis.

Protection against lethal sepsis following immunization with Candida species varies by isolate and inversely correlates with bone marrow tissue damage.

Protection against lethal Candida albicans (Ca)/Staphylococcus aureus (Sa) intra-abdominal infection (IAI)-mediated sepsis can be achieved by a novel form of trained innate immunity (TII) involving Gr-1+ myeloid-derived suppressor cells (MDSCs) that are induced by inoculation (immunization) with low virulence Candida species [i.e., Candida dubliniensis (Cd)] that infiltrate the bone marrow (BM). In contrast, more virulent Candida species (i.e., C. albicans), even at sub-lethal inocula, fail to induce similar levels of protection. The purpose of the present study was to test the hypothesis that the level of TII-mediated protection induced by Ca strains inversely correlates with damage in the BM as a reflection of virulence. Mice were immunized by intraperitoneal inoculation with several parental and mutant strains of C. albicans deficient in virulence factors (hyphal formation and candidalysin production), followed by an intraperitoneal Ca/Sa challenge 14 d later and monitored for sepsis and mortality. Whole femur bones were collected 24 h and 13 d after immunization and assessed for BM tissue/cellular damage via ferroptosis and histology. While immunization with standard but not sub-lethal inocula of most wild-type C. albicans strains resulted in considerable mortality, protection against lethal Ca/Sa IAI challenge varied by strain was usually less than that for C. dubliniensis, with no differences observed between parental and corresponding mutants. Finally, levels of protection afforded by the Ca strains were inversely correlated with BM tissue damage (R 2 = -0.773). TII-mediated protection against lethal Ca/Sa sepsis induced by Candida strain immunization inversely correlates with BM tissue/cellular damage as a reflection of localized virulence.

Current oral hygiene and recreational behavioral trends in HIV disease.

OBJECTIVE: HIV disease is evolving with more HIV+ persons experiencing a high quality of life with well-controlled viremia. We recently enrolled a large cohort of HIV+ and clinically relevant HIV- persons for oral microbiome analyses that included a questionnaire related to oral hygiene and recreational behaviors. Here, the questionnaire responses were analyzed for behavioral trends within the cohort, together with trends over time by comparison to a previous geographically centered HIV+ cohort. METHODS: Data were collected by questionnaire at baseline visits as cross-sectional assessments. Multivariable analyses were conducted for associations of HIV status as well as age, race, and sex, on oral hygiene/recreational behaviors. RESULTS: HIV+ subjects had reduced brushing frequency, but increased incidence of past cleanings and frequency of dry mouth, compared to the HIV- subjects. Within the entire cohort, positive associations were identified between age and several oral hygiene practices, and between age, race, and sex for several recreational behaviors. In comparison to the historical cohort, the contemporary HIV+ cohort participated in fewer high-risk behaviors, but with similar trends for smoking and oral hygiene practices. CONCLUSION: HIV status had little association with oral hygiene and recreational behaviors despite several differences in age, race, and sex. Behavioral trends over time support a higher quality of life in people currently living with HIV.

Effect of MMR Vaccination to Mitigate Severe Sequelae Associated With COVID-19: Challenges and Lessons Learned.

Mortality in COVID-19 cases was strongly associated with progressive lung inflammation and eventual sepsis. There is mounting evidence that live attenuated vaccines commonly administered during childhood, also provide beneficial non-specific immune effects, including reduced mortality and hospitalization due to unrelated infections. It has been proposed that live attenuated vaccine-associated non-specific effects are a result of inducing trained innate immunity to function more effectively against broader infections. In support of this, our laboratory has reported that immunization with a live attenuated fungal strain induces a novel form of trained innate immunity which provides protection against various inducers of sepsis in mice via myeloid-derived suppressor cells. Accordingly, we initiated a randomized control clinical trial with the live attenuated Measles, Mumps, Rubella (MMR) vaccine in healthcare workers in the greater New Orleans area aimed at preventing/reducing severe lung inflammation/sepsis associated with COVID-19 (ClinicalTrials.gov Identifier: NCT04475081). Included was an outcome to evaluate the myeloid-derived suppressor cell populations in blood between those administered the MMR vaccine vs placebo. The unanticipated emergency approval of several COVID-19 vaccines in the midst of the MMR clinical trials eliminated the ability to examine effects of the MMR vaccine on COVID-19-related health status. Unfortunately, we were also unable to show any impact of the MMR vaccine on peripheral blood myeloid-derived suppressor cells due to several inherent limitations (low percentages of blood leukocytes, small sample size), that also included a collaboration with a similar trial (CROWN CORONATION; ClinicalTrials.gov Identifier: NCT04333732) in St. Louis, MO. In contrast, monitoring the COVID-19 vaccine response in trial participants revealed that high COVID-19 antibody titers occurred more often in those who received the MMR vaccine vs placebo. While the trial was largely inconclusive, lessons learned from addressing several trial-associated challenges may aid future studies that test the non-specific beneficial immune effects of live attenuated vaccines.

Direct Comparison Between Intraoral Scanning and Alginate Impressions for Pediatric Patients: An In Vitro Study.

Purpose: To compare the accuracy and efficiency of both alginate and digital impression techniques in a method representative of a clinical setting. By evaluating the difference in fabrication time and accuracy for these techniques, it can be determined if using digital scanning is a viable alternative to alginate impressions to fabricate pediatric dental appliances.
Methods: Both the alginate and digital impressions were taken from the same pediatric typodont study model. The alginate impressions were handled and poured up, per product instructions, and digital impressions were 3D printed. A digital caliper was used to measure different landmarks on both the stone models and 3D printed models, and these measurements were compared to the pediatric typodont to determine accuracy. All steps in the process, including obtaining the impression, fabrication of the model and total time, were recorded to determine the efficiency of both methods.
Results: The alginate models had a significantly higher intercanine distance than the control. Measurements of anterior-posterior (AP) left, AP right, left central, and right central were larger than the control but did not show a significant difference in measurement. The alginate group had a significantly shorter impression time. Model fabrication time between the two groups was similar.
Conclusion: Alginate impressions require less chairside time and the digital technique was shown to be accurate in all measurements. Digital scanning may be a good alternative to alginate impressions for pediatric patients.

Independent Effects of HIV and Antiretroviral Therapy on the Oral Microbiome Identified by Multivariate Analyses.

The oral microbiome is an important predictor of health and disease. We recently reported significant yet modest effects of HIV under highly active antiretroviral therapy (ART) on the oral microbiome (bacterial and fungal) in a large cohort of HIV-positive (HIV+) and matched HIV-negative (HIV-) individuals. As it was unclear whether ART added to or masked further effects of HIV on the oral microbiome, the present study aimed to analyze the effects of HIV and ART independently, which also included HIV- subjects on preexposure prophylaxis (PrEP) therapy. Cross-sectional analyses of the effect of HIV devoid of ART (HIV+ ART- versus matched HIV- subjects) showed a significant effect on both the bacteriome and mycobiome (P < 0.024) after controlling for other clinical variables (permutational multivariate analysis of variance [PERMANOVA] of Bray-Curtis dissimilarity). Cross-sectional analyses evaluating the effects of ART (HIV+ ART+ versus HIV+ ART- subjects) revealed a significant effect on the mycobiome (P < 0.007) but not the bacteriome. In parallel longitudinal analyses, ART (before versus after the initiation of ART) had a significant effect on the bacteriome, but not the mycobiome, of HIV+ and HIV- PrEP subjects (P < 0.005 and P < 0.016, respectively). These analyses also revealed significant differences in the oral microbiome and several clinical variables between HIV- PrEP subjects (pre-PrEP) and the HIV-matched HIV- group (P < 0.001). At the species level, a small number of differences in both bacterial and fungal taxa were identified within the effects of HIV and/or ART. We conclude that the effects of HIV and ART on the oral microbiome are similar to those of the clinical variables but collectively are modest overall. IMPORTANCE The oral microbiome can be an important predictor of health and disease. For persons living with HIV (PLWH), HIV and highly active antiretroviral therapy (ART) may have a significant influence on their oral microbiome. We previously reported a significant effect of HIV with ART on both the bacteriome and mycobiome. It was unclear whether ART added to or masked further effects of HIV on the oral microbiome. Hence, it was important to evaluate the effects of HIV and ART independently. For this, multivariate cross-sectional and longitudinal oral microbiome analyses (bacteriome and mycobiome) were conducted within the cohort, including HIV+ ART+ subjects and HIV+ and HIV- (preexposure prophylaxis [PrEP]) subjects before and after the initiation of ART. While we report independent significant effects of HIV and ART on the oral microbiome, we conclude that their influence is similar to that of the clinical variables but collectively modest overall.

Bone Regeneration in Maxillary Sinus Augmentation Using Advanced Platelet-Rich Fibrin (A-PRF) and Plasma Rich in Growth Factors (PRGF): A Pilot Randomized Controlled Trial

The purpose of this pilot randomized controlled trial was to analyze and compare the effects of advanced platelet-rich fibrin (A-PRF) and plasma rich in growth factors (PRGF) combined with deproteinized bovine bone mineral (DBBM) on bone regeneration outcomes in maxillary sinus augmentation (MSA) procedures. A total of 15 patients in need of MSA were consecutively recruited. Maxillary sinuses were grafted with DBBM alone (control group), DBBM mixed with A-PRF (PRF group), or DBBM mixed with PRGF (PRGF group). After a 6-month healing period, bone core biopsy samples were collected prior to implant placement for histologic and histomorphometric analyses. The mean percentage of mineralized tissue (MT) was 20.33 ± 11.50 in the control group, 32.20 ± 7.29 for the PRF group, and 34.80 ± 6.83 for the PRGF group, with no statistically significant differences across the three groups (P > .05). The mean percentage of remaining bone grafting material (RBGM) was 24.00 ± 7.94 for the control group, 26.00 ± 7.78 for the PRF group, and 15.80 ± 8.23 for the PRGF group, with no statistically significant differences across the three groups (P > .05). Finally, the mean percentage of nonmineralized tissue (NMT) was 55.66 ± 7.77 for the control group, 41.40 ± 8.32 for the PRF group, and 49.60 ± 5.68 for the PRGF group, with no statistically signifcant differences across the three groups (P > .05). These findings suggest that the addition of A-PRF and PRGF to DBBM does not enhance new bone formation outcomes in maxillary sinus augmentation procedures. Neither of the two platelet concentrates were superior to the other in any of the variables assessed.

Structural and functional analysis of EntV reveals a 12 amino acid fragment protective against fungal infections.

Fungal pathogens are a continuing challenge due to few effective antifungals and a rise in resistance. In previous work, we described the inhibition of Candida albicans virulence following exposure to the 68 amino acid bacteriocin, EntV, secreted by Enterococcus faecalis. Here, to optimize EntV as a potential therapeutic and better understand its antifungal features, an X-ray structure is obtained. The structure consists of six alpha helices enclosing a seventh 16 amino acid helix (α7). The individual helices are tested for antifungal activity using in vitro and nematode infection assays. Interestingly, α7 retains antifungal, but not antibacterial activity and is also effective against Candida auris and Cryptococcus neoformans. Further reduction of α7 to 12 amino acids retains full antifungal activity, and excellent efficacy is observed in rodent models of C. albicans oropharyngeal, systemic, and venous catheter infections. Together, these results showcase EntV-derived peptides as promising candidates for antifungal therapeutic development.

Changes in concentrations of cervicovaginal immune mediators across the menstrual cycle: a systematic review and meta-analysis of individual patient data.

BACKGROUND: Hormonal changes during the menstrual cycle play a key role in shaping immunity in the cervicovaginal tract. Cervicovaginal fluid contains cytokines, chemokines, immunoglobulins, and other immune mediators. Many studies have shown that the concentrations of these immune mediators change throughout the menstrual cycle, but the studies have often shown inconsistent results. Our understanding of immunological correlates of the menstrual cycle remains limited and could be improved by meta-analysis of the available evidence. METHODS: We performed a systematic review and meta-analysis of cervicovaginal immune mediator concentrations throughout the menstrual cycle using individual participant data. Study eligibility included strict definitions of the cycle phase (by progesterone or days since the last menstrual period) and no use of hormonal contraception or intrauterine devices. We performed random-effects meta-analyses using inverse-variance pooling to estimate concentration differences between the follicular and luteal phases. In addition, we performed a new laboratory study, measuring select immune mediators in cervicovaginal lavage samples. RESULTS: We screened 1570 abstracts and identified 71 eligible studies. We analyzed data from 31 studies, encompassing 39,589 concentration measurements of 77 immune mediators made on 2112 samples from 871 participants. Meta-analyses were performed on 53 immune mediators. Antibodies, CC-type chemokines, MMPs, IL-6, IL-16, IL-1RA, G-CSF, GNLY, and ICAM1 were lower in the luteal phase than the follicular phase. Only IL-1α, HBD-2, and HBD-3 were elevated in the luteal phase. There was minimal change between the phases for CXCL8, 9, and 10, interferons, TNF, SLPI, elafin, lysozyme, lactoferrin, and interleukins 1ß, 2, 10, 12, 13, and 17A. The GRADE strength of evidence was moderate to high for all immune mediators listed here. CONCLUSIONS: Despite the variability of cervicovaginal immune mediator measurements, our meta-analyses show clear and consistent changes during the menstrual cycle. Many immune mediators were lower in the luteal phase, including chemokines, antibodies, matrix metalloproteinases, and several interleukins. Only interleukin-1α and beta-defensins were higher in the luteal phase. These cyclical differences may have consequences for immunity, susceptibility to infection, and fertility. Our study emphasizes the need to control for the effect of the menstrual cycle on immune mediators in future studies.

Methods Related to the Immunopathogenesis of Vulvovaginal Candidiasis and Associated Neutrophil Anergy.

Vulvovaginal candidiasis (VVC) is a common mucosal disease, caused primarily by Candida albicans that affects up to 75% of women of childbearing age. The pathogenesis of VVC and recurrent VVC (RVVC) is largely understood after decades of research. In this regard, an immunopathological response involving the migration of neutrophils that become dysfunctional (anergic) in the vaginal environment leads to the symptomatic conditions. However, immunotherapeutic strategies to correct the immunopathogenesis are still elusive. Much of the mechanistic discoveries have been uncovered using the established mouse model of chronic VVC. This chapter details the methods widely used for the mouse model of experimental VVC and associated outcome measures of the immunopathologic response and resulting symptomatic condition and focuses further on assays used to demonstrate "neutrophil anergy" in the model. These methods may serve as a source or resource for further experimentation with the ultimate goal to reduce or eliminate VVC/RVVC.

Efficacy of Candida dubliniensis and Fungal ß-Glucans in Inducing Trained Innate Immune Protection Against Inducers of Sepsis.

Fungal-bacterial intra-abdominal infections (IAI) can lead to sepsis with significant morbidity and mortality. We have established a murine model of Candida albicans (Ca) and Staphylococcus aureus (Sa) IAI that results in acute lethal sepsis. Prior intraperitoneal or intravenous inoculation with low virulence Candida dubliniensis (Cd) confers high level protection against lethal Ca/Sa IAI and sepsis. Protection via Cd immunization is associated with decreased pro-inflammatory cytokines and mediated by Gr-1+ putative myeloid-derived suppressor cells (MDSCs) representing a novel form of trained innate immunity (TII). The objective of these studies was to determine the extent of Cd-mediated TII against sepsis of broad origin and explore the potential of fungal cell wall components as abiotic immunogen alternatives to induce TII, including zymosan depleted of TLR2 activity (d-zymosan), or purified preparations of ß-glucan. Immunized mice were challenged 14 days post-immunization with a lethal array of live or abiotic inducers of sepsis, including Ca/Sa, Ca/Escherichia coli (Ca/Ec), LPS or untreated zymosan. Results showed that live Cd immunization was protective against sepsis induced by Ca/Ec and zymosan, but not LPS. Similar to protection against Ca/Sa, survival was dependent on Gr-1+ cells with no role for macrophages. Among the fungal cell wall compounds as immunogens, immunization with d-zymosan and an alkali-treated form of ß-glucan also resulted in significant protection against sepsis induced by Ca/Sa or Ca/Ec, but not LPS sepsis. Again, there was a strong dependence on Gr-1+ cells for protection with one exception, an added role for macrophages in the case of protection induced by alkali-treated ß-glucan. Overall, these results demonstrate that immunization with Cd as well as abiotic fungal cell components are capable of Gr-1+ cell-mediated trained innate immune protection against sepsis of broad microbial origin. In addition, abiotic ß-glucans represent potential alternatives to live Cd for protection against lethal polymicrobial sepsis.

Leukotrienes Are Dispensable for Vaginal Neutrophil Recruitment as Part of the Immunopathological Response During Experimental Vulvovaginal Candidiasis.

Recruitment of polymorphonuclear neutrophils (PMNs) into the vaginal lumen is the hallmark of an acute immunopathologic inflammatory response during vulvovaginal candidiasis (VVC) caused by Candida albicans. Recurrent VVC (RVVC) remains a chronic health burden in affected women worldwide despite the use of antifungal therapy. Based on the role leukotrienes (LTs) play in promoting inflammation, leukotriene receptor antagonists (LTRAs) targeted for LTB4 (etalocib) or LTC4, LTD4, and LTE4 (zafirlukast or montelukast) have been shown to reduce inflammation of epithelial tissues. An open-label pilot study using long-term regimens of zafirlukast in women with RVVC indicated the potential for some relief from recurrent episodes. To investigate this clinical observation further, we evaluated the effects of LT antagonistic agents and LT deficiency on the immunopathogenic response in a mouse model of VVC. Results showed that mice given daily intraperitoneal injections of individual LTRAs, starting 2days prior to vaginal inoculation with C. albicans and continuing through 14days post-inoculation, had no measurable reduction in PMN migration. The LTRAs were also ineffective in reducing levels of the hallmark vaginal inflammatory markers (S100A8, IL-1ß) and tissue damage (LDH) associated with the immunopathogenic response. Finally, LT-deficient 5-lipoxygenase knockout mice showed comparable levels of vaginal fungal burden and PMN infiltration to wild-type mice following inoculation with a vaginal (ATCC 96113) or laboratory (SC5314) C. albicans isolate. These results indicate that despite some clinical evidence suggestive of off-target efficacy of LTRAs in RVVC, LTs and associated signaling pathways appear to be dispensable in the immunopathogenesis of VVC.

A small molecule produced by Lactobacillus species blocks Candida albicans filamentation by inhibiting a DYRK1-family kinase.

The fungus Candida albicans is an opportunistic pathogen that can exploit imbalances in microbiome composition to invade its human host, causing pathologies ranging from vaginal candidiasis to fungal sepsis. Bacteria of the genus Lactobacillus are colonizers of human mucosa and can produce compounds with bioactivity against C. albicans. Here, we show that some Lactobacillus species produce a small molecule under laboratory conditions that blocks the C. albicans yeast-to-filament transition, an important virulence trait. It remains unexplored whether the compound is produced in the context of the human host. Bioassay-guided fractionation of Lactobacillus-conditioned medium linked this activity to 1-acetyl-ß-carboline (1-ABC). We use genetic approaches to show that filamentation inhibition by 1-ABC requires Yak1, a DYRK1-family kinase. Additional biochemical characterization of structurally related 1-ethoxycarbonyl-ß-carboline confirms that it inhibits Yak1 and blocks C. albicans biofilm formation. Thus, our findings reveal Lactobacillus-produced 1-ABC can prevent the yeast-to-filament transition in C. albicans through inhibition of Yak1.

Trained Innate Immunity Induced by Vaccination with Low-Virulence Candida Species Mediates Protection against Several Forms of Fungal Sepsis via Ly6G+ Gr-1+ Leukocytes.

We recently discovered a novel form of trained innate immunity (TII) induced by low-virulence Candida species (i.e., Candida dubliniensis) that protects against lethal fungal/bacterial infection. Mice vaccinated by intraperitoneal (i.p.) inoculation are protected against lethal sepsis following Candida albicans/Staphylococcus aureus (Ca/Sa) intra-abdominal infection (IAI) or Ca bloodstream infection (BSI). The protection against IAI is mediated by long-lived Gr-1+ leukocytes as putative myeloid-derived suppressor cells (MDSCs) and not by prototypical trained macrophages. This study aimed to determine if a similar TII mechanism (Gr-1+ cell-mediated suppression of sepsis) is protective against BSI and whether this TII can also be induced following intravenous (i.v.) vaccination. For this, mice were vaccinated with low-virulence Candida strains (i.p. or i.v.), followed by lethal challenge (Ca/Sa i.p. or Ca i.v.) 14 days later, and observed for sepsis (hypothermia, sepsis scoring, and serum cytokines), organ fungal burden, and mortality. Similar parameters were monitored following depletion of macrophages or Gr-1+ leukocytes during lethal challenge. The results showed that mice vaccinated i.p. or i.v. were protected against lethal Ca/Sa IAI or Ca BSI. In all cases, protection was mediated by Ly6G+ Gr-1+ putative granulocytic MDSCs (G-MDSCs), with no role for macrophages, and correlated with reduced sepsis parameters. Protection also correlated with reduced fungal burden in spleen and brain but not liver or kidney. These results suggest that Ly6G+ G-MDSC-mediated TII is induced by either the i.p. and i.v. route of inoculation and protects against IAI or BSI forms of systemic candidiasis, with survival correlating with amelioration of sepsis and reduced organ-specific fungal burden. IMPORTANCE Trained innate immunity (TII) is induced following immunization with live attenuated microbes and represents a clinically important strategy to enhance innate defenses. TII was initially demonstrated following intravenous inoculation with low-virulence Candida albicans, with protection against a subsequent lethal C. albicans intravenous bloodstream infection (BSI) mediated by monocytes with enhanced cytokine responses. We expanded this by describing a novel form of TII induced by intraperitoneal inoculation with low-virulence Candida that protects against lethal sepsis induced by polymicrobial intra-abdominal infection (IAI) via Gr-1+ leukocytes as putative myeloid-derived suppressor cells (MDSCs). In this study, we addressed these two scenarios and confirmed an exclusive role for Ly6G+ Gr-1+ leukocytes in mediating TII against either IAI or BSI via either route of inoculation, with protection associated with suppression of sepsis. These studies highlight the previously unrecognized importance of Ly6G+ MDSCs as central mediators of a novel form of TII termed trained tolerogenic immunity.

Effect of HIV/HAART and Other Clinical Variables on the Oral Mycobiome Using Multivariate Analyses.

The oral microbiome is considered an important factor in health and disease. We recently reported significant effects of HIV and several other clinical variables on the oral bacterial communities in a large cohort of HIV-positive and -negative individuals. The purpose of the present study was to similarly analyze the oral mycobiome in the same cohort. To identify fungi, the internal transcribed spacer 2 (ITS2) of the fungal rRNA genes was sequenced using oral rinse samples from 149 HIV-positive and 88 HIV-negative subjects that had previously undergone bacterial amplicon sequencing. Quantitative PCR was performed for total fungal content and total bacterial content. Interestingly, samples often showed predominance of a single fungal species with four major clusters predominated by Candida albicans, Candida dubliniensis, Malassezia restricta, or Saccharomyces cerevisiae Quantitative PCR analysis showed the Candida-dominated sample clusters had significantly higher total fungal abundance than the Malassezia or Saccharomyces species. Of the 25 clinical variables evaluated for potential influences on the oral mycobiome, significant effects were associated with caries status, geographical site of sampling, sex, HIV under highly active antiretroviral therapy (HAART), and missing teeth, in rank order of statistical significance. Investigating specific interactions between fungi and bacteria in the samples often showed Candida species positively correlated with Firmicutes or Actinobacteria and negatively correlated with Fusobacteria, Proteobacteria, and Bacteroidetes Our data suggest that the oral mycobiome, while diverse, is often dominated by a limited number of species per individual; is affected by several clinical variables, including HIV positivity and HAART; and shows genera-specific associations with bacterial groups.IMPORTANCE The oral microbiome is likely a key element of homeostasis in the oral cavity. With >600 bacterial species and >160 fungal species comprising the oral microbiome, influences on its composition can have an impact on both local and systemic health. We recently reported significant effects of HIV and several other clinical variables on the oral bacterial community in a large cohort of HIV-positive and -negative subjects. We describe here a comprehensive analysis of the oral mycobiome in the same cohort. Similar to the bacterial community, HIV under highly active antiretroviral therapy (HAART) had a significant impact on the mycobiome composition, but with less impact compared to other clinical variables. Additionally, unlike the oral bacterial microbiome, the oral mycobiome is often dominated by a single species with 4 major clusters of fungal communities. Together, these results suggest the oral mycobiome has distinct properties compared with the oral bacterial community, although both are equally impacted by HIV.