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Objective Helicobacter pyloriinfection is mostly a family-based infectious disease. To facilitate its prevention and management, a national consensus meeting was held to review current evidence and propose strategies for population-wide and family-basedH. pyloriinfection control and management to reduce the related disease burden. Methods Fifty-seven experts from 41 major universities and institutions in 20 provinces/regions of mainland China were invited to review evidence and modify statements using Delphi process and grading of recommendations assessment, development and evaluation system. The consensus level was defined as ≥80% for agreement on the proposed statements. Results Experts discussed and modified the original 23 statements on family-basedH. pyloriinfection transmission, control and management, and reached consensus on 16 statements. The final report consists of three parts: (1)H. pyloriinfection and transmission among family members, (2) prevention and management ofH. pyloriinfection in children and elderly people within households, and (3) strategies for prevention and management ofH. pyloriinfection for family members. In addition to the ‘test-and-treat’ and ‘screen-and-treat’ strategies, this consensus also introduced a novel third ‘family-basedH. pyloriinfection control and management’ strategy to prevent its intrafamilial transmission and development of related diseases. Conclusion H. pyloriis transmissible from person to person, and among family members. A family-basedH. pyloriprevention and eradication strategy would be a suitable approach to prevent its intra-familial transmission and related diseases. The notion and practice would be beneficial not only for Chinese residents but also valuable as a reference for other highly infected areas.

The roles of Candida albicans CHK1, a key gene from two-component system, in oral mucosal infection are not clear. This study evaluated the key roles of CHK1 gene in vitro and in vivo. The expression of CHK1 and its regulated virulence factors were tested during the oral epithelial cell infection. The production of lactate dehydrogenase, ROS, and IL-1α combined with the confocal and scanning electron microscope observation was employed to identify the capability of CHK1 in damaging the epithelial cells. Both immunocompetent and immunodeficient mice oropharyngeal infection models were involved to confirm the roles of CHK1 gene in vivo. The expression of CHK1 gene was significantly increased during the oral epithelial cell infection. The chk1Δ/Δ mutant failed to damage the epithelial cells or induce IL-α and ROS production. Interestingly, chk1Δ/Δ can also form the similar hyphae with WT and complementary strains. Accordingly, chk1Δ/Δ did not affect the adhesion and invasion rates of C. albicans to oral epithelial cells. However, chk1Δ/Δ significantly decreased the expression levels of the virulence factors, including ALS2, SAP6, and YWP1. The chk1Δ/Δ also failed to cause oral candidiasis in both immunocompetent and immunodeficient mice indicating that CHK1 gene from the two-component system is essential for the pathogenicity of C. albicans. KEY POINTS: • CHK1gene is essential for C. albicans in oral candidiasis • C. albicans without CHK1 gene can form "non-pathogenic" hyphae. • CHK1 gene regulates the virulence of C. albicans.

Background: Streptococcus pneumoniae is one of the most common pathogens to cause mucosal and invasive infection in humans. Most of the infection could be prevented through immunization by vaccines containing capsular polysaccharides but some infection may be caused by unencapsulated strains. Methods: Clinical isolates of S. pneumoniae from January 2012 to December 2015 at Chang Gung Memorial Hospital, Taiwan. Serotyping by PCR method was performed. Clinical and laboratory information of patients infected by non-typeable pneumococci (NTP) were collected and analyzed. Results: During the study period, 39 NTP isolates were identified. Most (21 of 39, 53.9%) were collected from purulent upper respiratory tract secretion. Others were from corneal abscess, sputum, and one from blood of a newborn. We recorded a 3.6-fold increase in the rate of isolation from 1.4% in 2012 to 5.0% in 2015 (p = 0.063). Co-infection was found in 24 cases; the major co-infecting pathogens included non-typeable Hemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus. Most (39 of 40, 97.5%) of the isolates were susceptible to both penicillin and ceftriaxone. The dominant sequence type ST1106 and an emerging sequence type ST7502 were recognized. Conclusions: A gradual increase of NTP infection was found in northern Taiwan in the pneumococcal conjugate vaccine era. Non-typeable pneumococci can cause respiratory and ophthalmological mucosal infection. Invasive infection can occur in newborns or young infants. Most of the isolates remained susceptible to penicillin and ceftriaxone. Keywords: Non-typeable Streptococcus pneumoniae, Pneumococcal conjugate vaccine, Penicillin, Ceftriaxone

The human pathogenic fungus Candida albicans is a frequent cause of mucosal infections. Although the ability to transition from the yeast to the hypha morphology is essential for virulence, hypha formation and host cell invasion per se are not sufficient for the induction of epithelial damage. Rather, the hypha-associated peptide toxin, candidalysin, a product of the Ece1 polyprotein, is the critical damaging factor. While synthetic, exogenously added candidalysin is sufficient to damage epithelial cells, the level of damage does not reach the same level as invading C. albicans hyphae. Therefore, we hypothesized that a combination of fungal attributes is required to deliver candidalysin to the invasion pocket to enable the full damaging potential of C. albicans during infection. Utilising a panel of C. albicans mutants with known virulence defects, we demonstrate that the full damage potential of C. albicans requires the coordinated delivery of candidalysin to the invasion pocket. This process requires appropriate epithelial adhesion, hyphal extension and invasion, high levels of ECE1 transcription, proper Ece1 processing and secretion of candidalysin. To confirm candidalysin delivery, we generated camelid V HHs (nanobodies) specific for candidalysin and demonstrate localization and accumulation of the toxin only in C. albicans-induced invasion pockets. In summary, a defined combination of virulence attributes and cellular processes is critical for delivering candidalysin to the invasion pocket to enable the full damage potential of C. albicans during mucosal infection. Take Aways: Candidalysin is a peptide toxin secreted by C. albicans causing epithelial damage. Candidalysin delivery to host cell membranes requires specific fungal attributes. Candidalysin accumulates in invasion pockets created by invasive hyphae. Camelid nanobodies enabled visualisation of candidalysin in the invasion pocket.

Mucosa protects the body against external pathogen invasion. However, pathogen colonies on the mucosa can invade the mucosa when the immunosurveillance is compromised, causing mucosal infection and subsequent diseases. Therefore, it is necessary to timely and effectively monitor and control pathogenic microorganisms through mucosal immunity.Candida albicansis the most prevalent fungi on the mucosa. TheC. albicanscolonies proliferate and increase their virulence, causing severe infectious diseases and even death, especially in immunocompromised patients. The normal host mucosal immune defense inhibits pathogenicC. albicansthrough stepwise processes, such as pathogen recognition, cytokine production, and immune cell phagocytosis. Herein, the current advances in the interactions betweenC. albicansand host mucosal immune defenses have been summarized to improve understanding on the immune mechanisms against fungal infections.

Rhinosporidiosis is a chronic mucosal infection caused by Rhinosporidium seeberi, an aquatic protistan parasite. It presents as nasal or ocular polypoidal or vascularized masses. It is endemic in tropical and subtropical areas, especially in South Asia; R. seeberi´s endemicity in the Americas is often overlooked. The objective of this study was to describe the demographic and clinical characteristics of patients with rhinosporidiosis in the Americas, its management, and patient outcomes. This study is a systematic review of cases of human rhinosporidiosis in the Americas reported in the literature from 1896 to February 28, 2019. This review screened 1,994 reports, of which 115 were eligible for further analysis. The selected reports described 286 cases of human rhinosporidiosis between 1896 and 2019. Cases were diagnosed in Brazil (32.2%), Colombia (24.4%), Paraguay (12.6%), and the United States (11.9%). The majority of the cases (91%) occurred in geographic areas with altitudes < 1,000 m above sea level and in areas with median temperatures ≥ 25°C (67.3%). Most of the patients presented nasal (65%) and ocular involvement (35%). Surgical treatment was provided for 99.6% of patients, but 19.8% of them recurred. This review describes the under-recognized geographic distribution and clinical presentation of rhinosporidiosis in the Americas and highlights clinical differences to cases in Asia, specifically in reference to a higher prevalence of ocular disease and higher relapse rates.

Oral carcinoma represents one of the most common malignancies worldwide. Oral squamous cell carcinomas (OSCCs) account over 90% of all oral malignant tumors and are characterized by high mortality in the advanced stages. Early diagnosis is often a challenge for its ambiguous appearance in early stages. Mucosal infection by the human papillomavirus (HPV) is responsible for a growing number of malignancies, particularly cervical cancer and oropharyngeal carcinomas. In addition, Candida albicans (C. albicans), which is the principal fungi involved in the oral cancer development, may induce carcinogenesis through several mechanisms, mainly promoting inflammation. Medical knowledge and research on adolescent/pediatric patients’ management and prevention are in continuous evolution. Besides, microbiota can play an important role in maintaining oral health and therefore all human health. The aim of this review is to evaluate epidemiological and pathophysiological characteristics of the several biochemical pathways involved during HPV and C. albicans infections in pediatric dentistry.

Trypanosoma cruzi is the intracellular parasite of Chagas disease, a chronic condition characterized by cardiac and gastrointestinal morbidity. Protective immunity requires CD4+ T cells, and Th1 cells and IFN-γ are important players in host defense. More recently, Th17 cells and IL-17 have been shown to exert protective functions in systemic T. cruzi infection. However, it remains unclear whether Th17 cells and IL-17A protect against mucosal infection, which is an important cause of human outbreaks. We found that IL-17RA knock-out (KO) mice are highly susceptible to orogastric infection, indicating an important function for this cytokine in mucosal immunity to T. cruzi. To investigate the specific role of Th17 cells for mucosal immunity, we reconstituted RAG1 KO mice with T. cruzi-specific T cell receptor transgenic Th17 cells prior to orogastric T. cruzi challenges. We found that Th17 cells provided protection against gastric mucosal T. cruzi infection, indicated by significantly lower stomach parasite burdens. In vitro macrophage infection assays revealed that protection by Th17 cells is reversed with IL-17A neutralization or loss of macrophage NADPH oxidase activity. Consistent with this, in vivo, mice lacking functional NADPH oxidase were not protected by Th17 cell transfer. These data are the first report that Th17 cells protect against mucosal T. cruzi infection, and identify a novel protective mechanism involving the induction of NADPH oxidase activity in macrophages by IL-17A. These studies provide important insights for Chagas vaccine development, and more broadly, increase our understanding of the diverse roles of Th17 cells in host defense.

Inducing humoral, cellular and mucosal immunity is likely to improve the effectiveness of HIV-1 vaccine strategies. Here, we tested a vaccine regimen in pigtail macaques using an intranasal (i.n.) recombinant Fowl Pox Virus (FPV)-gag pol env-IL-4R antagonist prime, intramuscular (i.m.) recombinant Modified Vaccinia Ankara Virus (MVA)-gag pol-IL-4R antagonist boost followed by an i.m SOSIP-gp140 boost. The viral vector—expressed IL-4R antagonist transiently inhibited IL-4/IL-13 signalling at the vaccination site. The SOSIP booster not only induced gp140-specific IgG, ADCC (antibody-dependent cellular cytotoxicity) and some neutralisation activity, but also bolstered the HIV-specific cellular and humoral responses. Specifically, superior sustained systemic and mucosal HIV Gag-specific poly-functional/cytotoxic CD4+ and CD8+ T cells were detected with the IL-4R antagonist adjuvanted strategy compared to the unadjuvanted control. In the systemic compartment elevated Granzyme K expression was linked to CD4+ T cells, whilst Granzyme B/TIA-1 to CD8+ T cells. In contrast, the cytotoxic marker expression by mucosal CD4+ and CD8+ T cells differed according to the mucosal compartment. This vector-based mucosal IL-4R antagonist/SOSIP booster strategy, which promotes cytotoxic mucosal CD4+ T cells at the first line of defence, and cytotoxic CD4+ and CD8+ T cells plus functional antibodies in the blood, may prove valuable in combating mucosal infection with HIV-1 and warrants further investigation.

Porcine epidemic diarrhea virus (PEDV) is an economically important pathogen that has evolved several mechanisms to evade type I IFN responses. Type III interferon (IFN-λ), an innate cytokine that primarily targets the mucosal epithelia, is critical in fighting mucosal infection in the host and has been reported to potently inhibit PEDV infection in vitro. However, how PEDV escapes IFN-λ antiviral response remains unclear. In this study, we found that PEDV infection induced significant IFN-λ expression in type I IFN-defective Vero E6 cells, but virus-induced endogenous IFN-λ did not reduce PEDV titers. Moreover, we demonstrated that PEDV escaped IFN-λ responses by substantially upregulating the suppressor of cytokine signaling protein 1 (SOCS1) expression, which impaired the induction of IFN-stimulated genes (ISGs) and dampened the IFN-λ antiviral response and facilitated PEDV replication in Vero E6 cells. We further showed that PEDV infection increased SOCS1 expression by decreasing host miR-30c-5p expression. MiR-30c-5p suppressed SOCS1 expression through targeting the 3′ untranslated region (UTR) of SOCS1. The inhibition of IFN-λ elicited ISGs expression by SOCS1 was specifically rescued by overexpression of miR-30c-5p. Collectively, our findings identify a new strategy by PEDV to escape IFN-λ-mediated antiviral immune responses by engaging the SOCS1/miR-30c axis, thus improving our understanding of its pathogenesis.

Background Vulvovaginal candidiasis (VVC) is a vaginal mucosal infection that usually affects women in their reproductive age. When the signs of VVC persist on a daily basis or last for a long time and repeat at least three times per year, the disease is considered chronic and recurrent. Objective The purpose of this study was to evaluate the frequency of HLA-DRB1 alleles in patients with recurrent vulvovaginal candidiasis (RVVC). Study design 120 patients with RVVC and 136 age-matched healthy controls underwent low-resolution HLA-DRB typing performed using the polymerase chain reaction-sequence-specific primers (PCR-SSP) technique. Results In the present work, we studied different genes that encode HLA-DRB (HLA-DRB1 / HLA-DRB3 / HLA-DRB4 / HLA-DRB5) and showed that HLA-DRB1×14, found in 25% of the patients. In the present study, the significant frequency of HLA-DRB1×10 in the control group suggests a resistant role of this allele to RVVC infections Conclusions In the HLA-DRB region, the DRB1×14 allele showed a higher frequency in the patients with RVVC than in the controls. Moreover, the higher frequency of DRB1×10 observed in the controls than in the patients with RVVC. These results demonstrate the HLA-DRB1 alleles are in relation with both susceptibility and immunity factors in RVVC infection and possible susceptible role of HLA-DRB1×14

Zika virus (ZIKV) is a major human pathogen. ZIKV can replicate in female and male reproductive organs, thus facilitating the human-human transmission cycle. Viral shedding in the semen can increase the risk of ZIKV transmission through sexual mode. Therefore, the vaginal and anorectal mucosa are relevant sites for ZIKV infection. However, the pathobiology of ZIKV transmission through the rectal route is not well understood. Here, we utilize a mouse model system to investigate the immunopathological consequences following ZIKV infection of the rectal mucosa compared to a subcutaneous route of infection. We show that ZIKV-rectal inoculation results in viremia with subclinical infection. ZIKV infects the mucosal epithelium and submucosal dendritic cells, inducing immune and inflammatory cell infiltration. Rectal transmission of ZIKV resulted in the generation of serum-neutralizing antibody responses. Mass cytometry analyses of splenocytes showed a significantly reduced level of inflammatory monocyte and neutrophil cellular responses in the rectal route group. Furthermore, immunological priming through the rectal mucosa with an attenuated ZIKV strain resulted in significant protection from lethal subcutaneous ZIKV challenge, further eliciting robust memory CD4-positive (CD4(+)) and CD8(+) T-cell and ZIKV-specific serum-neutralizing antibody responses. Thus, our study provides deeper immunopathobiological insights on rectal transmission and highlights a rational strategy for mucosal immunization. This model system recapitulates clinical aspects of human ZIKV disease outcome, where most infections are well controlled and result in subclinical and asymptomatic outcomes. IMPORTANCE Zika virus is a clinically significant human pathogen that is primarily transmitted and spread by Aedes species mosquitoes but is also sexually transmissible. The recent pandemic in the Americas led to an unprecedented increase of newborn babies with developmental brain and eye abnormalities. To date, there is no licensed vaccine or therapeutic intervention available for the fight against ZIKV. Understanding the sexual transmission of ZIKV through vaginal and rectal routes is necessary to restrict virus transmission and spread. This study examines the early immunological and pathological consequences of rectal and subcutaneous routes of ZIKV infection using a mouse model. We characterized the primary target cells of ZIKV infection and the subsequent mucosal immune responses to infection, and we demonstrate the protective effect of mucosal rectal immunization using an attenuated ZIKV strain. This mucosal vaccination approach can be further developed to prevent future ZIKV outbreaks.

This chapter mainly covers the microbes that cause mucosal infection. Many microorganisms can invade the oral mucosa, causing infectious stomatitis. Infectious stomatitis is classified as primary and secondary. The former refers to the inflammatory diseases that occur on the oral mucosa due to the direct invasion of pathogenic microorganisms, such as coccal stomatitis, necrotizing gingival stomatitis, and acute gangrenous stomatitis. The latter refers to the manifestation of systemic infection caused by pathogenic microorganisms in the oral mucosa, such as diphtheria and staphylococcal sepsis. Both primary and secondary infections can be divided into bacterial infection, viral infection, fungal infection, spirochetes infection, actinomycetes infection, mycoplasma infection, chlamydia infection, and rickettsial infection according to the different pathogenic microorganisms. The biological and pathogenic characteristics of representative Gram-positive bacteria, Gram-negative bacteria, mycoplasma, fungi, and virus were introduced in this chapter. Gram staining, plate culture, colony, and scanning electron microscope (SEM) images of each microorganism were also provided.

Th1 and Th17 cells have an established role in protective immunity to Bordetella pertussis, but this evidence is based largely on peripheral T cells. There is emerging evidence that local tissue-resident memory T (TRM) cells that accumulate in tissue following mucosal infection may be crucial for long-term immunity. In this study, we examined the role of respiratory CD4 TRM cells in immunity to B. pertussis. Natural immunity to B. pertussis induced by infection is considered long lasting and effective at preventing reinfection. Consistent with this, we found that convalescent mice rapidly cleared the bacteria after reinfection. Furthermore, CD4 T cells with a TRM cell phenotype (CD44+CD62L−CD69+ or CD44+CD62L−CD69+CD103+) accumulated in the lungs of mice during infection with B. pertussis and significantly expanded through local proliferation following reinfection. These CD4 TRM cells were B. pertussis specific and secreted IL-17 or IL-17 and IFN-γ. Treatment of mice with FTY720, which prevented migration of T and B cells from lymph nodes to the circulation, significantly exacerbated B. pertussis infection. This was associated with significantly reduced infiltration of central memory T cells and B cells into the lungs. However, the local expansion of TRM cells and the associated rapid clearance of the secondary infection were not affected by treatment with FTY720 before rechallenge. Moreover, adoptive transfer of lung CD4 TRM cells conferred protection in naive mice. Our findings reveal that Ag-specific CD4 TRM cells play a critical role in adaptive immunity against reinfection and memory induced by natural infection with B. pertussis.

Type III interferon-lambda (IFN-λ) plays a critical role against infection, particularly in mucosal infection in the respiratory and gastrointestinal tract. Our study and other previous studies have shown that porcine IFN-λ more efficiently curtails the infection of porcine epidemic diarrhea virus (PEDV) in the intestine epithelia than type I IFN, whereas IFN-λ3 exerts a more potent effect than IFN-λ1. However, the underlying mechanism remains elusive, and in particular, the transcriptional profile induced by IFN-λ3 has not been reported. Here, to resolve the mechanism responsible for the disparity between IFN-λ3 and type I IFN in anti-mucosal virus infection, we compared the transcription profiles induced by the two IFNs in porcine intestinal epithelial (IPEC-J2) cells by RNA-Seq. Our results showed that the pretreatment of IPEC-J2 cells with IFN-λ3 resulted in the differential expression of 983 genes. In contrast, IFN-α only modified the expression of 134 genes, and 110 of these genes were also observed in the response to IFN-λ3. A transcriptional enrichment analysis indicated that IFN-λ3 or IFN-α regulates multiple cellular processes and that IFN-λ3 activates more robust signaling pathways, particularly the antiviral Jak-STAT signaling pathway, than IFN-α. Furthermore, we verified the RNA-Seq results through an RT-qPCR analysis of IPEC-J2 cells and porcine enteroids. Moreover, transient expression of the porcine rsad2 and mx2 genes among the top 10 genes induced by IFN-λ3 significantly inhibited PEDV infection. Collectively, the data showed that IFN-λ3 induces a unique transcriptional profile that does not completely overlap with that induced by IFN-α and strongly elicits a set of genes responsible for the antiviral activity of IFN-λ3. These findings provide important knowledge regarding the elicited ISGs of type I and III IFNs in restricting porcine intestinal viral infection.

In modern medicine, physicians are frequently forced to balance immune suppression against immune stimulation to treat patients such as those undergoing transplants and chemotherapy. More-targeted therapies designed to preserve immunity and prevent opportunistic fungal infection in these patients could be informed by an understanding of how fungi interact with professional and nonprofessional immune cells in mucosal candidiasis. In this study, we intravitally imaged these host-pathogen dynamics during Candida infection in a transparent vertebrate model host, the zebrafish. Single-cell imaging revealed an unexpected partitioning of the inflammatory response between phagocytes and epithelial cells. Surprisingly, we found that in vivo cytokine profiles more closely match in vitro responses of epithelial cells rather than phagocytes. Furthermore, we identified a disconnect between canonical inflammatory cytokine production and phagocyte recruitment to the site of infection, implicating noncytokine chemoattractants. Our study contributes to a new appreciation for the specialization and cross talk among cell types during mucosal infection., Candida yeasts are common commensals that can cause mucosal disease and life-threatening systemic infections. While many of the components required for defense against Candida albicans infection are well established, questions remain about how various host cells at mucosal sites assess threats and coordinate defenses to prevent normally commensal organisms from becoming pathogenic. Using two Candida species, C. albicans and C. parapsilosis, which differ in their abilities to damage epithelial tissues, we used traditional methods (pathogen CFU, host survival, and host cytokine expression) combined with high-resolution intravital imaging of transparent zebrafish larvae to illuminate host-pathogen interactions at the cellular level in the complex environment of a mucosal infection. In zebrafish, C. albicans grows as both yeast and epithelium-damaging filaments, activates the NF-κB pathway, evokes proinflammatory cytokines, and causes the recruitment of phagocytic immune cells. On the other hand, C. parapsilosis remains in yeast morphology and elicits the recruitment of phagocytes without inducing inflammation. High-resolution mapping of phagocyte-Candida interactions at the infection site revealed that neutrophils and macrophages attack both Candida species, regardless of the cytokine environment. Time-lapse monitoring of single-cell gene expression in transgenic reporter zebrafish revealed a partitioning of the immune response during C. albicans infection: the transcription factor NF-κB is activated largely in cells of the swimbladder epithelium, while the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) is expressed in motile cells, mainly macrophages. Our results point to different host strategies for combatting pathogenic Candida species and separate signaling roles for host cell types.

Helicobacter pylori has coexisted with humans for at least 60.000 years and has evolved persistence strategies that allow it to evade host immunity and colonize its host for life. The VacA protein is expressed by all H. pylori strains and is required for high-level persistent infection in experimental mouse models. Here, we show that VacA targets myeloid cells in the gastric mucosa to create a tolerogenic environment that facilitates regulatory T-cell differentiation, while suppressing effector T-cell priming and functionality. Tregs that are induced in the periphery during H. pylori infection can be found not only in the stomach but also in the lungs of infected mice, where they are likely to affect immune responses to allergens., The gastric bacterium Helicobacter pylori causes a persistent infection that is directly responsible for gastric ulcers and gastric cancer in some patients and protective against allergic and other immunological disorders in others. The two outcomes of the Helicobacter-host interaction can be modeled in mice that are infected as immunocompetent adults and as neonates, respectively. Here, we have investigated the contribution of the Helicobacter immunomodulator VacA to H. pylori-specific local and systemic immune responses in both models. We found that neonatally infected mice are colonized at higher levels than mice infected as adults and fail to generate effector T-cell responses to the bacteria; rather, T-cell responses in neonatally infected mice are skewed toward Foxp3-positive (Foxp3+) regulatory T cells that are neuropilin negative and express RORγt. We found these peripherally induced regulatory T cells (pTregs) to be enriched, in a VacA-dependent manner, not only in the gastric mucosa but also in the lungs of infected mice. Pulmonary pTreg accumulation was observed in mice that have been infected neonatally with wild-type H. pylori but not in mice that have been infected as adults or mice infected with a VacA null mutant. Finally, we traced VacA to gastric lamina propria myeloid cells and show that it suppressed interleukin-23 (IL-23) expression by dendritic cells and induced IL-10 and TGF-β expression in macrophages. Taken together, the results are consistent with the idea that H. pylori creates a tolerogenic environment through its immunomodulator VacA, which skews T-cell responses toward Tregs, favors H. pylori persistence, and affects immunity at distant sites.

Vulvovaginal candidiasis (VVC) is a common mucosal infection caused by Candida spp., most frequently by Candida albicans, which may become recurrent and severely impacting the quality of life of susceptible women. Although it is increasingly being recognized that mucosal damage is mediated by an exaggerated inflammatory response, current therapeutic approaches are only based on antifungals that may relieve the symptomatology, but fail to definitely prevent recurrences. The unrestrained activation of the NLRP3 inflammasome with continuous production of IL-1β and recruitment of neutrophils is recognized as a pathogenic factor in VVC. We have previously shown that IL-22 is required to dampen pathogenic inflammasome activation in VVC via the NLRC4/IL-1Ra axis. However, IL-22 also regulates IL-18, a product of the inflammasome activity that regulates IL-22 expression. Here we describe a cross-regulatory circuit between IL-18 and IL-22 in murine VVC that is therapeutically druggable. We found that IL-18 production was dependent on IL-22 and NLRC4, and that IL-18, in turn, contributes to IL-22 activity. Like in IL-22 deficiency, IL-18 deficiency was associated with an increased susceptibility to VVC and unbalanced Th17/Treg response, suggesting that IL-18 can regulate both the innate and the adaptive responses to the fungus. Administration of the microbial metabolite indole-3-aldehyde, known to stimulate the production of IL-22 via the aryl hydrocarbon receptor (AhR), promoted IL-18 expression and protection against Candida infection. Should low levels of IL-18 be demonstrated in the vaginal fluids of women with recurrent VVC, targeting the AhR/IL-22/IL-18 pathway could be exploited for future therapeutic approaches in VVC. This study suggests that a deeper understanding of the mechanisms regulating inflammasome activity may lead to the identification of novel targets for intervention in VVC.

Oropharyngeal candidiasis is a common opportunistic mucosal infection of the oral cavity, mainly caused by an overgrowth of Candida albicans. This infection can inhibit nutritional intakes and strongly affect quality of life. To date, standard therapeutic strategies involving the administration of antifungal drugs can bring several side effects, not least the emergence of drug-resistant strains. The purpose of this study is to investigate the effectiveness of Saccharomyces cerevisiae CNCM I-3856 (live or inactivated cells) against oropharyngeal candidiasis. Our results show that administration of S. cerevisiae CNCM I-3856 (live or inactivated cells) in the oral cavity of C57BL/6J mice resulted in a protective effect against oropharyngeal candidiasis. The strongest effect was obtained with live S. cerevisiae CNCM I-3856. This was related to: (1) a decrease in C. albicans load in the oral cavity, esophagus, stomach, and duodenum; (2) an early resolution of inflammatory process in the tongue; (3) a marked reduction in C. albicans virulence factors; and (4) a consistent increase in neutrophil antimicrobial capacity. These findings suggest that S. cerevisiae products are potentially beneficial in the treatment of oropharyngeal candidiasis.

Candida albicansis a ubiquitous fungal symbiont that resides on diverse human barrier surfaces. Both mammalian and fungal cells can convert arachidonic acid into the lipid mediator, prostaglandin E2 (PGE2), but the physiological significance of fungal-derived PGE2remains elusive. Here we report that aC. albicansmutant deficient in PGE2production suffered a loss of competitive fitness in the murine gastrointestinal (GI) tract and that PGE2supplementation mitigated this fitness defect. Impaired fungal PGE2production affected neither thein vitrofitness ofC. albicansnor hyphal morphogenesis and virulence in either systemic or mucosal infection models. Fungus-derived PGE2improved intra-GI fitness ofC. albicansby diminishing the killing ofC. albicansby phagocytes. Consequently, ablation of colonic phagocytes abrogated the fitness boost conferred by fungal PGE2. These observations suggest thatC. albicanshas evolved the capacity to produce PGE2from arachidonic acid, a host-derived precursor, to promote its own colonization of the host gut. Analogous mechanisms might undergird host-microbe interactions of other symbiont fungi.Author SummaryCandida albicansis a symbiont fungus that resides in the gut of a majority of people without provoking disease. However, residentC. albicanscan bloom and turn pathogenic in a subset of individuals who are immunocompromised due to infections or chemotherapy or who suffer a disruption of their intestinal microbial community due to antibiotic use. However, the fungal and host factors that regulate the fitness ofC. albicansas a symbiont or an invasive pathogen remain poorly understood. Here we focused on the physiological role of fungus-derived prostaglandin E2 (PGE2) in the fitness ofC. albicansusing a PGE2-deficientC. albicansstrain and mouse models of infections and intestinal symbiosis. We found that fungal PGE2, contrary to previously described functions of promoting virulence, played no role in fungal pathogenicityin vivo. Instead, fungal PGE2specifically augmented the ability ofC. albicansto colonize the gut, in part by reducing fungal killing by intestinal phagocytes. Our results suggest that fungal PGE2synthetic pathways may be prophylactically targeted in individuals susceptible to invasive infections.

In the absence of an active prophylactic vaccine against HIV-1, passively administered, broadly neutralizing antibodies (bnAbs) identified in some chronically infected persons were shown to prevent HIV-1 infection in animal models. However, passive administration of bnAbs may not be suited to prevent sexual HIV-1 transmission in high-risk cohorts, as a continuous high level of active bnAbs may be difficult to achieve at the primary site of sexual transmission, the human vagina with its acidic pH. Therefore, we used Lactobacillus, a natural commensal in the healthy vaginal microbiome, to express bn nanobodies (VHH) against HIV-1 that we reported previously. After demonstrating that recombinant VHHA6 expressed in E. coli was able to protect humanized mice from mucosal infection by HIV-1Bal, we expressed VHHA6 in a soluble or in a cell-wall-anchored form in Lactobacillus rhamnosus DSM14870. This strain is already clinically applied for treatment of bacterial vaginosis. Both forms of VHHA6 neutralized a set of primary epidemiologically relevant HIV-1 strains in vitro. Furthermore, VHHA6 was still active at an acidic pH. Thus, lactobacilli expressing bn VHH potentially represent an attractive vector for the passive immunization of women in cohorts at high risk of HIV-1 transmission.

Cytolytic proteins and peptide toxins are classical virulence factors of several bacterial pathogens which disrupt epithelial barrier function, damage cells and activate or modulate host immune responses. Such toxins have not been identified previously in human pathogenic fungi. Here we identify the first, to our knowledge, fungal cytolytic peptide toxin in the opportunistic pathogen Candida albicans. This secreted toxin directly damages epithelial membranes, triggers a danger response signalling pathway and activates epithelial immunity. Membrane permeabilization is enhanced by a positive charge at the carboxy terminus of the peptide, which triggers an inward current concomitant with calcium influx. C. albicans strains lacking this toxin do not activate or damage epithelial cells and are avirulent in animal models of mucosal infection. We propose the name 'Candidalysin' for this cytolytic peptide toxin; a newly identified, critical molecular determinant of epithelial damage and host recognition of the clinically important fungus, C. albicans.

Brucellosis remains a significant zoonotic threat worldwide. Humans and animals acquire infection via their oropharynx and upper respiratory tract following oral or aerosol exposure. After mucosal infection, brucellosis develops into a systemic disease. Mucosal vaccination could offer a viable alternative to conventional injection practices to deter disease. Using a nasal vaccination approach, the ΔznuA B. melitensis was found to confer potent protection against pulmonary Brucella challenge, and reduce colonization of spleens and lungs by more than 2500-fold, with >50% of vaccinated mice showing no detectable brucellae. Furthermore, 10-fold more brucellae-specific, interferon-γ (IFN-γ)-producing CD8(+) T cells than CD4(+) T cells were induced in the spleen and respiratory lymph nodes. Evaluation of pulmonary and splenic CD8(+) T cells from mice vaccinated with ΔznuA B. melitensis revealed that these expressed an activated effector memory (CD44(hi)CD62L(lo)CCR7(lo)) T cells producing elevated levels of IFN-γ, tumor necrosis factor-α, perforin and granzyme B. To assess the relative importance of these increased numbers of CD8(+) T cells, CD8(-/-) mice were challenged with virulent B. melitensis, and they showed markedly increased bacterial loads in organs in contrast to similarly challenged CD4(-/-) mice. Only ΔznuA B. melitensis- and Rev-1-vaccinated CD4(-/-) and wild-type mice, not CD8(-/-) mice, were completely protected against Brucella challenge. Determination of cytokines responsible for conferring protection showed the relative importance of IFN-γ, but not interleukin-17 (IL-17). Unlike wild-type (wt) mice, IL-17 was greatly induced in IFN-γ(-/-) mice, but IL-17 could not substitute for IFN-γ's protection, although an increase in brucellae dissemination was observed upon in vivo IL-17 neutralization. These results show that nasal ΔznuA B. melitensis vaccination represents an attractive means to stimulate systemic and mucosal immune protection via CD8(+) T-cell engagement.

West Nile virus, Kunjin strain (WNVKUN) is endemic in Northern Australia, but rarely causes clinical disease in humans and horses. Recently, WNVKUN genomic material was detected in cutaneous lesions of farmed saltwater crocodiles (Crocodylus porosus), but live virus could not be isolated, begging the question of the pathogenesis of these lesions. Crocodile hatchlings were experimentally infected with either 105 (n = 10) or 104 (n = 11) TCID50-doses of WNVKUN and each group co-housed with six uninfected hatchlings in a mosquito-free facility. Seven hatchlings were mock-infected and housed separately. Each crocodile was rotationally examined and blood-sampled every third day over a 3-week period. Eleven animals, including three crocodiles developing typical skin lesions, were culled and sampled 21 days post-infection (dpi). The remaining hatchlings were blood-sampled fortnightly until experimental endpoint 87 dpi. All hatchlings remained free of overt clinical disease, apart from skin lesions, throughout the experiment. Viremia was detected by qRT-PCR in infected animals during 2&ndash, 17 dpi and in-contact animals 11&ndash, 21 dpi, indicating horizontal mosquito-independent transmission. Detection of viral genome in tank-water as well as oral and cloacal swabs, collected on multiple days, suggests that shedding into pen-water and subsequent mucosal infection is the most likely route. All inoculated animals and some in-contact animals developed virus-neutralizing antibodies detectable from 17 dpi. Virus-neutralizing antibody titers continued to increase in exposed animals until the experimental endpoint, suggestive of persisting viral antigen. However, no viral antigen was detected by immunohistochemistry in any tissue sample, including from skin and intestine. While this study confirmed that infection of saltwater crocodiles with WNVKUN was associated with the formation of skin lesions, we were unable to elucidate the pathogenesis of these lesions or the nidus of viral persistence. Our results nevertheless suggest that prevention of WNVKUN infection and induction of skin lesions in farmed crocodiles may require management of both mosquito-borne and water-borne viral transmission in addition to vaccination strategies.

The past four decades have seen a staggering escalation in the number of invasive fungal infections worldwide.[...].

The hyphal development of Candida albicans (C. albicans) has been considered as an essential virulent factor for host cell damage. However, the missing link between hyphae and virulence of C. albicans is also been discovered. Here, we identified that the null mutants of ERG3 and ERG11, two key genes in ergosterol biosynthesis pathway, can form typical hyphae but failed to cause the oral mucosal infection in vitro and in vivo for the first time. In particular, the erg3Δ/Δ and erg11Δ/Δ strains co-cultured with epithelial cells significantly reduced the adhesion, damage, and cytokine (interleukin-1α (IL-1α)) production, whereas the invasion was not affected in vitro. Importantly, they were incapable of extensive hyphal invasion, formation of micro-abscesses, and tongue epithelium damage compared to wild type due to the decrease of the colonization and epithelial infection area in a murine oropharyngeal candidiasis model. The fluconazole (FLC), an antifungal targeted at ergosterol biosynthesis, relieved the epithelial infection of C. albicansin vitro and in vivo even under non-growth inhibitory dosage confirming the virulent contribution of ergosterol biosynthesis pathway. The erg3Δ/Δ and erg11Δ/Δ strains were cleared by macrophages similar to wild type, whereas their virulence factors including agglutinin-like sequence 1 (Als1), secreted aspartyl proteinase 6 (Sap6), and hyphal wall protein-1 (Hwp1) were significantly reduced indicated that the non-toxicity might not result from the change on immune tolerance but the defective virulence. The incapacity of erg3Δ/Δ and erg11Δ/Δ in epithelial infection highlights the contribution of ergosterol biosynthesis pathway to C. albicans pathogenesis and fluconazole can not only eliminate the fungal pathogens but also reduced their virulence even at low dosage.

The fungus Candida albicans thrives on a variety of human mucosae, yet the fungal determinants that contribute to fitness on these surfaces remain underexplored. Here, by screening a collection of C. albicans deletion strains in a mouse model of oral infection (oropharyngeal candidiasis), we identify several novel regulatory genes that modulate the fitness of the fungus in this locale. We investigate in detail the interplay between the host mucosa and one of the identified mutants and establish that the C. albicans transcription regulator CUP9 is a key determinant of mucosal colonisation. Deletion of cup9 resulted in the formation of more foci of colonisation and heightened persistence in infected tongues. Furthermore, the cup9 mutant produced longer and denser filaments in the oral mucosa without eliciting an enhanced local immune response. Consistent with its role in oral colonisation, we show that CUP9's top target of regulation is a major effector of Candida's adherence to buccal cells. Finally, we establish that CUP9 also governs the interplay of the fungus with vaginal epithelial cells and has a role in vaginal infections, another common mucosal disease associated with Candida. Thus, our findings reveal a mechanism whereby C. albicans can regulate proliferation on mucosal surfaces.

Chlamydia pecorum is a mucosal infection, which causes debilitating disease of the urinary tract, reproductive tract and ocular sites of koalas (Phascolarctos cinereus). While antibiotics are available for treatment, they are detrimental to the koalas’ gastrointestinal tract microflora leaving the implementation of a vaccine as an ideal option for the long-term management of koala populations. We have previously reported on the successes of an anti-chlamydial recombinant major outer membrane protein (rMOMP) vaccine however, recombinant protein based vaccines are not ideal candidates for scale up from the research level to small-medium production level for wider usage. Peptide based vaccines are a promising area for vaccine development, because peptides are stable, cost effective and easily produced. In this current study, we assessed, for the first time, the immune responses to a synthetic peptide based anti-chlamydial vaccine in koalas. Five healthy male koalas were vaccinated with two synthetic peptides derived from C. pecorum MOMP and another five healthy male koalas were vaccinated with full length recombinant C. pecorum MOMP (genotype G). Systemic (IgG) and mucosal (IgA) antibodies were quantified and pre-vaccination levels compared to post-vaccination levels (12 and 26 weeks). MOMP-peptide vaccinated koalas produced Chlamydia-specific IgG and IgA antibodies, which were able to recognise not only the genotype used in the vaccination, but also MOMPs from several other koala C. pecorum genotypes. In addition, IgA antibodies induced at the ocular site not only recognised recombinant MOMP protein but also, whole native chlamydial elementary bodies. Interestingly, some MOMP-peptide vaccinated koalas showed a stronger and more sustained vaccine-induced mucosal IgA antibody response than observed in MOMP-protein vaccinated koalas. These results demonstrate that a synthetic MOMP peptide based vaccine is capable of inducing a Chlamydia-specific antibody response in koalas and is a promising candidate for future vaccine development.

Polymicrobial infections often include both fungi and bacteria and can complicate patient treatment and resolution of infection. Cross-kingdom interactions among bacteria, fungi, and/or the immune system during infection can enhance or block virulence mechanisms and influence disease progression. The fungus Candida albicans and the bacterium Pseudomonas aeruginosa are coisolated in the context of polymicrobial infection at a variety of sites throughout the body, including mucosal tissues such as the lung. In vitro , C. albicans and P. aeruginosa have a bidirectional and largely antagonistic relationship. Their interactions in vivo remain poorly understood, specifically regarding host responses in mediating infection. In this study, we examine trikingdom interactions using a transparent juvenile zebrafish to model mucosal lung infection and show that C. albicans and P. aeruginosa are synergistically virulent. We find that high C. albicans burden, fungal epithelial invasion, swimbladder edema, and epithelial extrusion events serve as predictive factors for mortality in our infection model. Longitudinal analyses of fungal, bacterial, and immune dynamics during coinfection suggest that enhanced morbidity is associated with exacerbated C. albicans pathogenesis and elevated inflammation. The P. aeruginosa quorum-sensing-deficient Δ lasR mutant also enhances C. albicans pathogenicity in coinfection and induces extrusion of the swimbladder. Together, these observations suggest that C. albicans-P. aeruginosa cross talk in vivo can benefit both organisms to the detriment of the host.

Mouse model is an appropriate tool for pathogenic determination and study of host defenses during the fungal infection. Here, we established a mouse model of candidiasis with concurrent oral and vaginal mucosal infection. Two C. albicans strains sourced from clinical candidemia (SC5314) and mucosal infection (ATCC62342) were tested in ICR mice. The different combinational panels covering estrogen and immunosuppressive agents, cortisone, prednisolone and cyclophosphamide were used for concurrent oral and vaginal candidiasis establishment. Prednisolone in combination with estrogen proved an optimal mode for concurrent mucosal infection establishment. The model maintained for 1 week with fungal burden reached at least 10(5) cfu/g of tissue. This mouse model was evaluated by in vivo pharmacodynamics of fluconazole and host mucosal immunity of IL-17 and IL-23. Mice infected by SC5314 were cured by fluconazole. An increase in IL-23 in both oral and vaginal homogenates was observed after infection, while IL-17 only had a prominent elevation in oral tissue. This model could properly mimic complicated clinical conditions and provides a valuable means for antifungal assay in vivo and may also provide a useful method for the evaluation of host-fungal interactions.

Understanding the pathogenic mechanisms of Mycobacterium avium subspecies paratuberculosis (MAP) and the host responses to Johne's disease is complicated by the multi-faceted disease progression, late-onset host reaction and the lack of available ex vivo infection models. We describe a novel cell culture passage model that mimics the course of infection in vivo. The developed model simulates the interaction of MAP with the intestinal epithelial cells, followed by infection of macrophages and return to the intestinal epithelium. MAP internalization triggers a minimal inflammatory response. After passage through a macrophage phase, bacterial reinfection of MDBK epithelial cells, representing the late phase of intestinal mucosal infection, is associated with increased synthesis of the pro-inflammatory transcripts of IL-6, CCL5, IL-8 and IL-18, paired with decreased levels of TGFβ. Transcriptome analysis of MAP from each stage of epithelial cell infection identified increased expression of lipid biosynthesis and lipopeptide modification genes in the inflammatory phenotype of MAP. Total lipid analysis by HPLC-ES/MS indicates different lipidomic profiles between the two phenotypes and a unique set of lipids composing the inflammatory MAP phenotype. The presence of selected upregulated lipid-modification gene transcripts in samples of ileal tissue from cows diagnosed with Johne's disease supports and validates the model. By using the relatively simple cell culture passage model, we show that MAP alters its lipid composition during intracellular infection and acquires a pro-inflammatory phenotype, which likely is associated with the inflammatory phase of Johne's disease.

Candida albicans belongs to the normal microbial flora on epithelial surfaces of humans. However, under certain, still not fully understood conditions, it can become pathogenic and cause a spectrum of diseases, from local infections to life-threatening septicemia. We investigated a panel of antimicrobial proteins and peptides (AMPs), potentially involved in mucosal immunity against this pathogen. Out of six studied AMPs, psoriasin was most up-regulated during a mucosal infection, an acute episode of recurrent Candida vulvovaginitis, although candidacidal activity has not been demonstrated. We here show that psoriasin binds to β-glucan, a basic component of the C. albicans cell wall, and thereby inhibits adhesion of the pathogen to surfaces and increases IL-8 production by mucosal epithelial cells. In conclusion, we show a novel mechanism of action of psoriasin. By inhibiting C. albicans adhesion and by enhancing cytokine production, psoriasin contributes to the immune response against C. albicans. Key messages The antimicrobial peptide psoriasin is highly up-regulated during a local mucosal infection, Candida albicans vulvovaginitis.Psoriasin binds to β-glucan in the Candida albicans cell wall and thereby inhibits adhesion of the pathogen.Binding of psoriasin to Candida albicans induces an immune response by mucosal epithelial cells. Electronic supplementary material The online version of this article (10.1007/s00109-018-1637-6) contains supplementary material, which is available to authorized users.

Zika virus is present in urine, saliva, tears, and breast milk, but the transmission risk associated with these body fluids is currently unknown. Here we evaluate the risk of Zika virus transmission through mucosal contact in rhesus macaques. Application of high-dose Zika virus directly to the tonsils of three rhesus macaques results in detectable plasma viremia in all animals by 2 days post-exposure; virus replication kinetics are similar to those observed in animals infected subcutaneously. Three additional macaques inoculated subcutaneously with Zika virus served as saliva donors to assess the transmission risk from contact with oral secretions from an infected individual. Seven naive animals repeatedly exposed to donor saliva via the conjunctivae, tonsils, or nostrils did not become infected. Our results suggest that there is a risk of Zika virus transmission via the mucosal route, but that the risk posed by oral secretions from individuals with a typical course of Zika virus infection is low., Zika virus (ZIKV) is present in body fluids, including saliva, but transmission risk through mucosal contact is not well known. Here, the authors show that oropharyngeal mucosal infection of macaques with a high ZIKV dose results in viremia, but that transmission risk from saliva of infected animals is low.

Due to the increasing emergence of antibiotic-resistant strains of enteropathogenic bacteria, development of alternative treatments to fight against gut infections is a major health issue. While vaccination requires that a proper combination of antigen, adjuvant, and delivery route is defined to elicit protective immunity at mucosae, oral delivery of directly active antibody preparations, referred to as passive immunization, sounds like a valuable alternative. Along the gut, the strategy suffers, however, from the difficulty to obtain sufficient amounts of antibodies with the appropriate specificity and molecular structure for mucosal delivery. Physiologically, at the antibody level, the protection of gastrointestinal mucosal surfaces against enteropathogens is principally mediated by secretory IgA and secretory IgM. We previously demonstrated that purified human plasma-derived IgA and IgM can be associated with secretory component to generate biologically active secretory-like IgA and IgM (SCIgA/M) that can protect epithelial cells from infection by Shigella flexneri in vitro. In this study, we aimed at evaluating the protective potential of these antibody preparations in vivo. We now establish that such polyreactive preparations bind efficiently to Salmonella enterica Typhimurium and trigger bacterial agglutination, as observed by laser scanning confocal microscopy. Upon delivery into a mouse ligated intestinal loop, SCIgA/M-mediated aggregates persist in the intestinal environment and limit the entry of bacteria into intestinal Peyer’s patches via immune exclusion. Moreover, oral administration to mice of immune complexes composed of S. Typhimurium and SCIgA/M reduces mucosal infection, systemic dissemination, and local inflammation. Altogether, our data provide valuable clues for the future appraisal of passive oral administration of polyreactive plasma-derived SCIgA/M to combat infection by a variety of enteropathogens.

The intestinal tract provides ideal niches for several different microbial species, which are collectively called the gut microbiota. A key host immune effector that controls the microbiota and prevents mucosal infection is IgA. Gut microbiota-derived factors are largely classified into molecular pattern recognition receptor ligands and nutrient-derived metabolites including short-chain fatty acids and adenosine triphosphate. Along with host-derived factors such as retinoic acid, various cytokines and cytokine-like molecules, gut microbial products profoundly shape B cell responses. Gut microbial products can directly regulate B cell activation and differentiation. They can also indirectly affect B cells through epithelial cells, T cells, and myeloid cell subsets. We highlight the various direct and indirect mechanisms by which microbial products regulate humoral immunity.

Objective Antimicrobial peptides (AMP) provide protection from infection by pathogenic microorganisms and restrict bacterial growth at epithelial surfaces to maintain mucosal homeostasis. In addition, they exert a significant anti-inflammatory activity. Here we analysed the anatomical distribution and biological activity of an orally administered AMP in the context of bacterial infection and host–microbial homeostasis. Design The anatomical distribution as well as antibacterial and anti-inflammatory activity of the endogenous AMP cryptdin 2 and the synthetic peptide Pep19-2.5 at the enteric mucosal surface were analysed by immunostaining, functional viability and stimulation assays, an oral Salmonella enterica subsp. enterica sv. Typhimurium (S. Typhimurium) model and comparative microbiota analysis. Results Endogenous cryptdin 2 was found attached to bacteria of the enteric microbiota within the intestinal mucus layer. Similarly, the synthetic peptide Pep19-2.5 attached rapidly to bacterial cells, exhibited a marked affinity for the intestinal mucus layer in vivo, altered the structural organisation of endotoxin in a mucus matrix and demonstrated potent anti-inflammatory and antibacterial activity. Oral Pep19-2.5 administration induced significant changes in the composition of the enteric microbiota as determined by high-throughput 16S rDNA sequencing. This may have contributed to the only transient improvement of the clinical symptoms after oral infection with S. Typhimurium. Conclusions Our findings demonstrate the anti-inflammatory activity and mucus affinity of the synthetic AMP Pep19-2.5 and characterise the influence on microbiota composition and enteropathogen infection after oral administration.

Summary Candida albicans and streptococci of the mitis group colonize the oral cavities of the majority of healthy humans. While C. albicans is considered an opportunistic pathogen, streptococci of this group are broadly considered avirulent or even beneficial organisms. However, recent evidence suggests that multi-species biofilms with these organisms may play detrimental roles in host homeostasis and may promote infection. In this review we summarize the literature on molecular interactions between members of this streptococcal group and C. albicans, with emphasis on their potential role in the pathogenesis of opportunistic oral mucosal infections.

The human body is home to countless microorganisms that can modulate the transition between health and disease. When conditions in the host favor the growth of pathogens, the populations can shift toward their dominance and/or cause disbyosis. In the mouth, commensal organisms are typically most abundant, but when oral hygiene is neglected and sugar is frequently consumed and/or there is a disruption in saliva production/flow, organisms associated with dental caries disease eg, Streptococcus mutans (S. mutans) become more prevalent. These organisms interact with dietary sugars and host saliva to form complex 3-dimensional biofilms on pellicle-coated teeth. The production of exopolysaccharides (eg, via S. mutans-sucrose interactions) modulates the assembly of the biofilm matrix, while acid production (eg, by acidogenic flora) and low pH within the biofilm facilitate the demineralization of the adjacent tooth enamel. During the assembly of cariogenic biofilms, S. mutans likely interacts and competes with other oral microbial species in the mouth. Although it is widely recognized that bacterial-fungal interactions commonly occur on mucosal surfaces, their possible role in dental caries has received limited attention. This review provides evidence that interactions between S. mutans and Candida albicans (C. albicans) may be involved in the pathogenesis of early childhood caries.

Flexible adaptation to the host environment is a critical trait that underpins the success of numerous microbes. The polymorphic fungus Candida albicans has evolved to persist in the numerous challenging niches of the human body. The interaction of C. albicans with a mucosal surface is an essential prerequisite for fungal colonisation and epitomises the complex interface between microbe and host. C. albicans exhibits numerous adaptations to a healthy host that permit commensal colonisation of mucosal surfaces without provoking an overt immune response that may lead to clearance. Conversely, fungal adaptation to impaired immune fitness at mucosal surfaces enables pathogenic infiltration into underlying tissues, often with devastating consequences. This review will summarise our current understanding of the complex interactions that occur between C. albicans and the mucosal surfaces of the human body.

Many viruses invade mucosal surfaces to establish infection in the host. Some viruses are restricted to mucosal surfaces, whereas others disseminate to sites of secondary replication. Studies of strain-specific differences in reovirus mucosal infection and systemic dissemination have enhanced an understanding of viral determinants and molecular mechanisms that regulate viral pathogenesis. After peroral inoculation, reovirus strain type 1 Lang replicates to high titers in the intestine and spreads systemically, whereas strain type 3 Dearing (T3D) does not. These differences segregate with the viral S1 gene segment, which encodes attachment protein σ1 and nonstructural protein σ1s. In this study, we define genetic determinants that regulate reovirus-induced pathology following intranasal inoculation and respiratory infection. We report that two laboratory isolates of T3D, T3D C and T3D F , differ in the capacity to replicate in the respiratory tract and spread systemically; the T3D C isolate replicates to higher titers in the lungs and disseminates, while T3D F does not. Two nucleotide polymorphisms in the S1 gene influence these differences, and both S1 gene products are involved. T3D C amino acid polymorphisms in the tail and head domains of σ1 protein influence the sensitivity of virions to protease-mediated loss of infectivity. The T3D C polymorphism at nucleotide 77, which leads to coding changes in both S1 gene products, promotes systemic dissemination from the respiratory tract. A σ1s-null virus produces lower titers in the lung after intranasal inoculation and disseminates less efficiently to sites of secondary replication. These findings provide new insights into mechanisms underlying reovirus replication in the respiratory tract and systemic spread from the lung.

NanoString nCounter is a recently developed platform that can make direct multiplexed measurement of gene expression using color-coded probe pairs (Geiss et al., Nat Biotechnol 26(3):317-325, 2008; Malkov et al., BMC Res Notes 2:80, 2009). We have found that this platform is uniquely suitable for quantification of pathogen gene expression during infection, where pathogen RNA comprises a tiny portion of total RNA isolated from the infected tissue. Here, we describe a protocol that we have successfully applied to a number of pathogens across multiple infection models, including both invasive and mucosal infection by Candida albicans, and lung infection by Aspergillus fumigatus and Cryptococcus neoformans.

Mycobacterium bovis bacilli Calmette-Guerin (BCG) is used as a benchmark to compare the immunogenicity of new vaccines against tuberculosis. This live vaccine is administered intradermal, but several new studies show that changing the route to mucosal immunisation represents an improved strategy. We analysed the immunomodulatory functions of BCG on human neutrophils and primary airway epithelial cells (AECs), as the early events of mucosal immune activation are unclear. Neutrophils and the primary epithelial cells were found to express the IL-17A receptor subunit IL-17RA, while the expression of IL-17RE was only observed on epithelial cells. BCG stimulation specifically reduced neutrophil IL-17RA and epithelial IL-17RE expression. BCG induced neutrophil extracellular traps (NETs), but did not have an effect on apoptosis as measured by transcription factor forkhead box O3 (FOXO3). BCG stimulation of AECs induced CXCL8 secretion and neutrophil endothelial passage towards infected epithelia. Infected epithelial cells and neutrophils were not found to be a source of IL-17 cytokines or the interstitial collagenase MMP-1. However, the addition of IFNγ or IL-17A to BCG stimulated primary epithelial cells increased epithelial IL-6 secretion, while the presence of IFNγ reduced neutrophil recruitment. Using our model of mucosal infection we revealed that BCG induces selective mucosal innate immune responses that could lead to induction of vaccine-mediated protection of the lung.

IL-12 is a cytokine with links to both innate and adaptive immunity systems. In mice, its deletion leads to acute susceptibility to oral infection with the yeastCandida albicans, whereas such mice are resistant to systemic disease. However, it is an essential component of the adaptive response that leads to the generation of Th1-type cytokine responses and protection against disseminated disease. This paper presents an overview of the role of IL-12 in models of systemic and mucosal infection and the possible relationships between them.

A plethora of intricate and dynamic molecular interactions occur between microbes and the epithelial cells that form the mucosal surfaces of the human body. Fungi, particularly species of Candida, are commensal members of our microbiota, continuously interacting with epithelial cells. Transient and localised perturbations to the mucosal environment can facilitate the overgrowth of fungi, causing infection. This minireview will examine the direct and indirect mechanisms by which Candida species and epithelial cells interact with each other, and explore the factors involved in the central processes of adhesion, invasion, and destruction of host mucosal surfaces.

The impact of antibiotics on the host's protective microbiota and the resulting increased susceptibility to mucosal infection are poorly understood. In this study, antibiotic regimens commonly applied to murine enteritis models are used to examine the impact of antibiotics on the intestinal microbiota, the time course of recovery of the biota, and the resulting susceptibility to enteric Salmonella infection. Molecular analysis of the microbiota showed that antibiotic treatment has an impact on the colonization of the murine gut that is site and antibiotic dependent. While combinations of antibiotics were able to eliminate culturable bacteria, none of the antibiotic treatments were effective at sterilizing the intestinal tract. Recovery of total bacterial numbers occurs within 1 week after antibiotic withdrawal, but alterations in specific bacterial groups persist for several weeks. Increased Salmonella translocation associated with antibiotic pretreatment corrects rapidly in association with the recovery of the most dominant bacterial group, which parallels the recovery of total bacterial numbers. However, susceptibility to intestinal colonization and mucosal inflammation persists when mice are infected several weeks after withdrawal of antibiotics, correlating with subtle alterations in the intestinal microbiome involving alterations of specific bacterial groups. These results show that the colonizing microbiotas are integral to mucosal host protection, that specific features of the microbiome impact different aspects of enteric Salmonella pathogenesis, and that antibiotics can have prolonged deleterious effects on intestinal colonization resistance.

We have developed a murine cytomegalovirus (MCMV)-vectored vaccine expressing the mouse zona-pellucida-3 gene (rMCMV-ZP3), which successfully induces infertility in experimentally inoculated laboratory or wild-derived mice. However, the future success of this vector as a fully disseminating vaccine in free-living mice may be compromised by pre-existing immunity since there is a high prevalence of naturally acquired MCMV infection in these mice. To evaluate the effect of prior immunity to MCMV on vaccine efficacy, we constructed two new biologically effective recombinant MCMV vectors expressing the mouse ZP3 protein from two MCMV strains (N1 and G4) derived from free-living mice. In wild mice, mixed MCMV infection is common and could be acquired either by simultaneous coinfection or sequential infection with different MCMV strains. Interestingly, while coinfection with both wild-type and rMCMV-ZP3 via the intraperitoneal route reduced the impact of the rMCMV-ZP3, prior infection with the same wild-type strain as that used to construct the rMCMV-ZP3 abrogated the immunocontraceptive effects of either N1-ZP3 or G4-ZP3. However, prior infection with G4 28 days before the introduction of N1-ZP3 had a reduced influence on the efficacy of the rMCMV-ZP3. Thus, the strain of virus and the timing of prior infection are factors that may influence the efficacy of the rMCMV-ZP3. Given that mixed infection of mice with MCMV is common, it is possible that prior immunity acquired by natural mucosal infection may have less a less inhibitory effect on the immunocontraceptive outcome.

Rodent models of oral, vaginal and gastrointestinal Candida infection are described and discussed in terms of their scientific merits. The common feature of all experimental mucosal Candida infections is the need for some level of host immunocompromise or exogenous treatment to ensure quantitatively reproducible disease. A growing literature describes the contributions of such candidiasis models to our understanding of certain aspects of fungal virulence and host response to mucosal Candida albicans challenge. Evidence to date shows that T-lymphocyte responses dominate host immune defences to oral and gastrointestinal challenge, while other, highly compartmentalized responses defend vaginal surfaces. By contrast the study of C. albicans virulence factors in mucosal infection models has only begun to unravel the complex of attributes required to define the difference between strongly and weakly muco-invasive strains.

Numerous studies have demonstrated that targeting Ag to Fc receptors (FcR) on APCs can enhance humoral and cellular immunity. However, studies are lacking that examine both the use of FcR-targeting in generating immune protection against infectious agents and the use of FcRs in the induction of mucosal immunity. Francisella tularensis is a category A intracellular mucosal pathogen. Thus, intense efforts are underway to develop a vaccine against this organism. We hypothesized that protection against mucosal infection with F. tularensis would be significantly enhanced by targeting inactivated F. tularensis live vaccine strain (iFt) to FcRs at mucosal sites, via intranasal immunization with mAb-iFt complexes. These studies demonstrate for the first time that: 1) FcR-targeted immunogen enhances immunogen-specific IgA production and protection against subsequent infection in an IgA-dependent manner, 2) FcγR and neonatal FcR are crucial to this protection, and 3) inactivated F. tularensis, when targeted to FcRs, enhances protection against the highly virulent SchuS4 strain of F. tularensis, a category A biothreat agent. In summary, these studies show for the first time the use of FcRs as a highly effective vaccination strategy against a highly virulent mucosal intracellular pathogen.

Oral candidiasis is an opportunistic infection caused primarily by Candida albicans. However, in recent years, species of non-albicans Candida have been implicated more frequently in mucosal infection. Candida species usually reside as commensal organisms and are part of normal oral microflora. Determining exactly how transformation from commensal to pathogen takes place and how it can be prevented is continuous challenge for clinical doctors. Candidal adherence to mucosal surfaces is considered as a critical initial step in the pathogenesis of oral candidiasis. Acrylic dentures, acting as reservoirs, play an important role in increasing the risk from Candida colonisation. Thus, this review discusses what is currently known about the adhesion of non-albicans Candida species of oral origin to buccal epithelial cells and denture acrylics.

The mucosal immune system represents the first line of defense against Brucella infection in nature. We used genetically deficient mice to identify the lymphocytes and signaling pathways implicated in the control of primary and secondary intranasal infection with B. melitensis. Our analysis of primary infection demonstrated that the effectors implicated differ at the early and late stages and are dependent on the organ. TCR-δ, TAP1, and IL-17RA deficiency specifically affects early control of Brucella in the lungs, whereas MHC class II (MHCII) and IFN-γR deficiency impairs late control in the lungs, spleen, and liver. Interestingly, IL-12p35−/− mice display enhanced Brucella growth in the spleen but not in the lungs or liver. Secondary intranasal infections are efficiently contained in the lung. In contrast to an i.p. infectious model, in which IL-12p35, MHCII, and B cells are strictly required for the control of secondary infection, we observed that only TCR-β deficiency or simultaneous neutralization of IL-12p35– and IL-17A–dependent pathways impairs the memory protective response against a secondary intranasal infection. Protection is not affected by TCR-δ, MHCII, TAP1, B cell, IL-17RA, or IL-12p35 deficiency, suggesting that CD4+ and CD8+ α/β+ T cells are sufficient to mount a protective immune response and that an IL-17A–mediated response can compensate for the partial deficiency of an IFN-γ–mediated response to control a Brucella challenge. These findings demonstrate that the nature of the protective memory response depends closely on the route of infection and highlights the role of IFN-γ–and IL-17RA–mediated responses in the control of mucosal infection by Brucella.