Role of Sequencing the Measles Virus Hemagglutinin Gene and Hypervariable Region in the Measles Outbreak Investigations in Sweden During 2013-2014.

INTRODUCTION: It is increasingly difficult to differentiate measles viruses (MeVs) relating to certain outbreaks on the basis of the nucleoprotein (N) gene sequence only, as the diversity of circulating MeV strains has decreased. We studied genomic regions that could provide better molecular discrimination between epidemiologically linked and unlinked MeV variants identified in Sweden during 2013-2014. METHODS: The hemagglutinin (H) gene and hypervariable region between the fusion and matrix genes (MF-HVR) from 53 MeV-positive samples were amplified and sequenced. Data on phylogenetic clustering of MeVs on the basis of N, H, and MF-HVR sequences were compared to epidemiological data. RESULTS: MeVs were genotyped: 27 were B3, and 26 were D8. One genotype B3 cluster based on the N gene sequence contained epidemiologically unrelated viruses from 4 outbreaks, whereas analysis of H and MF-HVR sequences separated them into phylogenetic clusters consistent with the epidemiological data. Similarly, the single cluster of viruses with a genotype D8 N gene could be divided into the 5 outbreak groups on the basis of the phylogeny of MF-HVR sequences. CONCLUSIONS: A detailed picture of MeV circulation with more-defined links between outbreaks was obtained by sequencing the H gene and MF-HVR. Further identification and better genetic characterization of MeVs internationally is essential in identifying sources and routes of MeV spread within and beyond Europe in the elimination end game.

Seroprevalence of SARS-CoV-2 in Sweden, April 26 to May 9, 2021.

A national point seroprevalence study of SARS-CoV-2 was conducted in Sweden in April-May 2021. In total, 2860 individuals 3 to 90 years old from a probability-based web panel were included. Results showed that an estimated 32.6% of the population in Sweden had detectable levels of antibodies, and among non-vaccinated 20.1% had detectable levels of antibodies. We tested for differences in seroprevalence between age groups and by sex and estimated seroprevalence among previously infected participants by time since reporting.

Unphosphorylated CsgD controls biofilm formation in Salmonella enterica serovar Typhimurium.

The transcriptional regulator CsgD of Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major regulator of biofilm formation required for the expression of csgBA, which encodes curli fimbriae, and adrA, coding for a diguanylate cyclase. CsgD is a response regulator with an N-terminal receiver domain with a conserved aspartate (D59) as a putative target site for phosphorylation and a C-terminal LuxR-like helix-turn-helix DNA binding motif, but the mechanisms of target gene activation remained unclear. To study the DNA-binding properties of CsgD we used electrophoretic mobility shift assays and DNase I footprint analysis to show that unphosphorylated CsgD-His(6) binds specifically to the csgBA and adrA promoter regions. In vitro transcription analysis revealed that CsgD-His(6) is crucial for the expression of csgBA and adrA. CsgD-His(6) is phosphorylated by acetyl phosphate in vitro, which decreases its DNA-binding properties. The functional impact of D59 in vivo was demonstrated as S. Typhimurium strains expressing modified CsgD protein (D59E and D59N) were dramatically reduced in biofilm formation due to decreased protein stability and DNA-binding properties in the case of D59E. In summary, our findings suggest that the response regulator CsgD functions in its unphosphorylated form under the conditions of biofilm formation investigated in this study.

Bistable expression of CsgD in biofilm development of Salmonella enterica serovar typhimurium.

Bacterial persistence in the environment and in the infected host is often aided by the formation of exopolymer-enclosed communities known as biofilms. Heterogeneous gene expression takes place in microcompartments formed within the complex biofilm structure. This study describes cell differentiation within an isogenic bacterial cell population based on the example of biofilm formation by Salmonella enterica serovar Typhimurium. We analyzed the expression of the major biofilm regulator CsgD at the single-cell level with a chromosomal CsgD-green fluorescent protein (GFP) translational fusion. In individual cells, CsgD-GFP expression is mostly found in the cytoplasm. Quantitative expression analysis and results from three different models of S. Typhimurium biofilms demonstrated that CsgD is expressed in a bistable manner during biofilm development. CsgD expression is, however, monomodal when CsgD is expressed in larger amounts due to a promoter mutation or elevated levels of the secondary signaling molecule c-di-GMP. High levels of CsgD-GFP are associated with cellular aggregation in all three biofilm models. Furthermore, the subpopulation of cells expressing large amounts of CsgD is engaged in cellulose production during red, dry, and rough (rdar) morphotype development and in microcolony formation under conditions of continuous flow. Consequently, bistability at the level of CsgD expression leads to a corresponding pattern of task distribution in S. Typhimurium biofilms.

A role for the EAL-like protein STM1344 in regulation of CsgD expression and motility in Salmonella enterica serovar Typhimurium.

The bacterial second messenger cyclic di-GMP (c-di-GMP) regulates the transition between sessility and motility. In Salmonella enterica serovar Typhimurium, the expression of CsgD, the regulator of multicellular rdar morphotype behavior, is a major target of c-di-GMP signaling. CsgD expression is positively regulated by at least two diguanylate cyclases, GGDEF domain proteins, and negatively regulated by at least four phosphodiesterases, EAL domain proteins. Here, we show that in contrast to EAL domain proteins acting as phosphodiesterases, the EAL-like protein STM1344 regulated CsgD expression positively and motility negatively. STM1344, however, did not have a role in c-di-GMP turnover and also did not bind the nucleotide. STM1344 acted upstream of the phosphodiesterases STM1703 and STM3611, previously identified to participate in CsgD downregulation, where it repressed their expression. Consequently, although STM1344 has not retained a direct role in c-di-GMP metabolism, it still participates in the regulation of c-di-GMP turnover and has a role in the transition between sessility and motility.

Identifying infection-associated genes of Candida albicans in the postgenomic era.

The human pathogenic yeast Candida albicans can cause an unusually broad range of infections reflecting a remarkable potential to adapt to various microniches within the human host. The exceptional adaptability of C. albicans is mediated by rapid alterations in gene expression in response to various environmental stimuli and this transcriptional flexibility can be monitored with tools such as microarrays. Using such technology it is possible to (1) capture a genome-wide portrait of the transcriptome that mirrors the environmental conditions, (2) identify known genes, signalling pathways and transcription factors involved in pathogenesis, (3) identify new patterns of gene expression and (4) identify previously uncharacterized genes that may be associated with infection. In this review, we describe the molecular dissection of three distinct stages of infections, covering both superficial and invasive disease, using in vitro, ex vivo and in vivo infection models and microarrays.

Effectiveness of influenza vaccine against influenza A in Europe in seasons of different A(H1N1)pdm09 and the same A(H3N2) vaccine components (2016-17 and 2017-18).

INTRODUCTION: Influenza A(H3N2) viruses predominated in Europe in 2016-17. In 2017-18 A(H3N2) and A(H1N1)pdm09 viruses co-circulated. The A(H3N2) vaccine component was the same in both seasons; while the A(H1N1)pdm09 component changed in 2017-18. In both seasons, vaccine seed A(H3N2) viruses developed adaptations/alterations during propagation in eggs, impacting antigenicity. METHODS: We used the test-negative design in a multicentre primary care case-control study in 12 European countries to measure 2016-17 and 2017-18 influenza vaccine effectiveness (VE) against laboratory-confirmed influenza A(H1N1)pdm09 and A(H3N2) overall and by age group. RESULTS: During the 2017-18 season, the overall VE against influenza A(H1N1)pdm09 was 59% (95% CI: 47-69). Among those aged 0-14, 15-64 and ≥65 years, VE against A(H1N1)pdm09 was 64% (95% CI: 37-79), 50% (95% CI: 28-66) and 66% (95% CI: 42-80), respectively. Overall VE against influenza A(H3N2) was 28% (95% CI: 17-38) in 2016-17 and 13% (95% CI: -15 to 34) in 2017-18. Among 0-14-year-olds VE against A(H3N2) was 28% (95%CI: -10 to 53) and 29% (95% CI: -87 to 73), among 15-64-year-olds 34% (95% CI: 18-46) and 33% (95% CI: -3 to 56) and among those aged ≥65 years 15% (95% CI: -10 to 34) and -9% (95% CI: -74 to 32) in 2016-17 and 2017-18, respectively. CONCLUSIONS: Our study suggests the new A(H1N1)pdm09 vaccine component conferred good protection against circulating strains, while VE against A(H3N2) was <35% in 2016-17 and 2017-18. The egg propagation derived antigenic mismatch of the vaccine seed virus with circulating strains may have contributed to this low effectiveness. A(H3N2) seed viruses for vaccines in subsequent seasons may be subject to the same adaptations; in years with lower than expected VE, recommendations of preventive measures other than vaccination should be given in a timely manner.

2015/16 I-MOVE/I-MOVE+ multicentre case-control study in Europe: Moderate vaccine effectiveness estimates against influenza A(H1N1)pdm09 and low estimates against lineage-mismatched influenza B among children.

BACKGROUND: During the 2015/16 influenza season in Europe, the cocirculating influenza viruses were A(H1N1)pdm09 and B/Victoria, which was antigenically distinct from the B/Yamagata component in the trivalent influenza vaccine. METHODS: We used the test-negative design in a multicentre case-control study in twelve European countries to measure 2015/16 influenza vaccine effectiveness (VE) against medically attended influenza-like illness (ILI) laboratory-confirmed as influenza. General practitioners swabbed a systematic sample of consulting ILI patients and a random sample of influenza-positive swabs was sequenced. We calculated adjusted VE against influenza A(H1N1)pdm09, A(H1N1)pdm09 genetic group 6B.1 and influenza B overall and by age group. RESULTS: We included 11 430 ILI patients, of which 2272 were influenza A(H1N1)pdm09 and 2901 were influenza B cases. Overall VE against influenza A(H1N1)pdm09 was 32.9% (95% CI: 15.5-46.7). Among those aged 0-14, 15-64 and ≥65 years, VE against A(H1N1)pdm09 was 31.9% (95% CI: -32.3 to 65.0), 41.4% (95% CI: 20.5-56.7) and 13.2% (95% CI: -38.0 to 45.3), respectively. Overall VE against influenza A(H1N1)pdm09 genetic group 6B.1 was 32.8% (95% CI: -4.1 to 56.7). Among those aged 0-14, 15-64 and ≥65 years, VE against influenza B was -47.6% (95% CI: -124.9 to 3.1), 27.3% (95% CI: -4.6 to 49.4) and 9.3% (95% CI: -44.1 to 42.9), respectively. CONCLUSIONS: Vaccine effectiveness (VE) against influenza A(H1N1)pdm09 and its genetic group 6B.1 was moderate in children and adults, and low among individuals ≥65 years. Vaccine effectiveness (VE) against influenza B was low and heterogeneous among age groups. More information on effects of previous vaccination and previous infection is needed to understand the VE results against influenza B in the context of a mismatched vaccine.

Exploring the effect of previous inactivated influenza vaccination on seasonal influenza vaccine effectiveness against medically attended influenza: Results of the European I-MOVE multicentre test-negative case-control study, 2011/2012-2016/2017.

BACKGROUND: Results of previous influenza vaccination effects on current season influenza vaccine effectiveness (VE) are inconsistent. OBJECTIVES: To explore previous influenza vaccination effects on current season VE among population targeted for vaccination. METHODS: We used 2011/2012 to 2016/2017 I-MOVE primary care multicentre test-negative data. For each season, we compared current season adjusted VE (aVE) between individuals vaccinated and unvaccinated in previous season. Using unvaccinated in both seasons as a reference, we then compared aVE between vaccinated in both seasons, current only, and previous only. RESULTS: We included 941, 2645 and 959 influenza-like illness patients positive for influenza A(H1N1)pdm09, A(H3N2) and B, respectively, and 5532 controls. In 2011/2012, 2014/2015 and 2016/2017, A(H3N2) aVE point estimates among those vaccinated in previous season were -68%, -21% and -19%, respectively; among unvaccinated in previous season, these were 33%, 48% and 46%, respectively (aVE not computable for influenza A(H1N1)pdm09 and B). Compared to current season vaccination only, VE for both seasons' vaccination was (i) similar in two of four seasons for A(H3N2) (absolute difference [ad] 6% and 8%); (ii) lower in three of four seasons for influenza A(H1N1)pdm09 (ad 18%, 26% and 29%), in two seasons for influenza A(H3N2) (ad 27% and 39%) and in two of three seasons for influenza B (ad 26% and 37%); (iii) higher in one season for influenza A(H1N1)pdm09 (ad 20%) and influenza B (ad 24%). CONCLUSIONS: We did not identify any pattern of previous influenza vaccination effect. Prospective cohort studies documenting influenza infections, vaccinations and vaccine types are needed to understand previous influenza vaccinations' effects.

Recommendations for enterovirus diagnostics and characterisation within and beyond Europe.

Enteroviruses (EV) can cause severe neurological and respiratory infections, and occasionally lead to devastating outbreaks as previously demonstrated with EV-A71 and EV-D68 in Europe. However, these infections are still often underdiagnosed and EV typing data is not currently collected at European level. In order to improve EV diagnostics, collate data on severe EV infections and monitor the circulation of EV types, we have established European non-polio enterovirus network (ENPEN). First task of this cross-border network has been to ensure prompt and adequate diagnosis of these infections in Europe, and hence we present recommendations for non-polio EV detection and typing based on the consensus view of this multidisciplinary team including experts from over 20 European countries. We recommend that respiratory and stool samples in addition to cerebrospinal fluid (CSF) and blood samples are submitted for EV testing from patients with suspected neurological infections. This is vital since viruses like EV-D68 are rarely detectable in CSF or stool samples. Furthermore, reverse transcriptase PCR (RT-PCR) targeting the 5'noncoding regions (5'NCR) should be used for diagnosis of EVs due to their sensitivity, specificity and short turnaround time. Sequencing of the VP1 capsid protein gene is recommended for EV typing; EV typing cannot be based on the 5'NCR sequences due to frequent recombination events and should not rely on virus isolation. Effective and standardized laboratory diagnostics and characterisation of circulating virus strains are the first step towards effective and continuous surveillance activities, which in turn will be used to provide better estimation on EV disease burden.

[Polio close to eradication]. / Polio närmar sig utrotning.

Polio close to eradication The WHO Global Polio Eradication Initiative has been highly successful. With a dramatic decrease in polio since it started in 1988, the number of globally reported cases reached a record low of 37 in 2016. This article describes the WHO Endgame Strategic Plan including milestones that have been reached and challenges that have to be overcome in order to reach the goal of polio eradication by 2020. Efforts to strengthen immunizations systems and to detect and interrupt polio virus transmission focus on the three remaining endemic countries, namely Pakistan, Afghanistan and Nigeria. In 2016 the WHO took the first step to withdraw the oral polio vaccine (OPV) by globally shifting from trivalent to bivalent OPV.  The role of the inactivated vaccine (IPV) in the final phase of eradication and in the post-eradication situation is also considered. Certification of eradication and containment of all polio virus by the end of 2020 is a key objective. Legacy planning includes mainstreaming polio functions into ongoing public health programmes.

Increased Expression of Toll-Like Receptors 4, 5, and 9 in Small Bowel Mucosa from Patients with Irritable Bowel Syndrome.

The aim of our study was to compare patients with irritable bowel syndrome (IBS) and healthy controls regarding the expression of toll-like receptors 2, 4, 5, and 9 (TLR2, TLR4, TLR5, and TLR9), the primary mucosal receptors of bacterial components, in small and large bowel mucosa. Methods. We analysed biopsies from jejunum and sigmoid colon of 22 patients (17 females) with IBS aged 18-66 (median: 39) years and 14 healthy volunteers (12 females) aged 22-61 (median: 42) years. Eight patients had constipation-predominant IBS (C-IBS), 7 had diarrhoea-predominant IBS (D-IBS), and 7 had IBS without predominance of constipation or diarrhoea. We analysed mRNA levels for TLRs using quantitative PCR and distribution of TLRs in mucosa using immunohistochemistry. Results. We found increased mRNA expression of TLR4 (mean fold change 1.85 ± 0.31 versus 1.0 ± 0.20; p < 0.05), TLR5 (1.96 ± 0.36 versus 1.0 ± 0.20; p < 0.05) and TLR9 (2.00 ± 0.24 versus 1.0 ± 0.25; p < 0.01) but not of TLR2 in the small bowel mucosa from patients with IBS compared to the controls. There was no significant difference in mRNA levels for TLRs in colon mucosa between patients and controls. Conclusion. Upregulation of TLR4, TLR5, and TLR9 suggests the involvement of bacteria or dysregulation of the immune response to commensal flora in small bowel mucosa in IBS patients.

No difference in small bowel microbiota between patients with irritable bowel syndrome and healthy controls.

Several studies have indicated that colonic microbiota may exhibit important differences between patients with irritable bowel syndrome (IBS) and healthy controls. Less is known about the microbiota of the small bowel. We used massive parallel sequencing to explore the composition of small bowel mucosa-associated microbiota in patients with IBS and healthy controls. We analysed capsule biopsies from the jejunum of 35 patients (26 females) with IBS aged 18-(36)-57 years and 16 healthy volunteers (11 females) aged 20-(32)-48 years. Sequences were analysed based on taxonomic classification. The phyla with the highest total abundance across all samples were: Firmicutes (43%), Proteobacteria (23%), Bacteroidetes (15%), Actinobacteria (9.3%) and Fusobacteria (7.0%). The most abundant genera were: Streptococcus (19%), Veillonella (13%), Prevotella (12%), Rothia (6.4%), Haemophilus (5.7%), Actinobacillus (5.5%), Escherichia (4.6%) and Fusobacterium (4.3%). We found no difference among major phyla or genera between patients with IBS and controls. We identified a cluster of samples in the small bowel microbiota dominated by Prevotella, which may represent a common enterotype of the upper small intestine. The remaining samples formed a gradient, dominated by Streptococcus at one end and Escherichia at the other.

Human enteroendocrine cell responses to infection with Chlamydia trachomatis: a microarray study.

BACKGROUND: Enteroendocrine cells (EEC) are highly specialized cells producing signalling molecules vital to the normal functions of the gut. Recently, we showed altered protein distribution in Chlamydia infected EEC in vitro. The aim of this study was to perform a microarray analysis of the response pattern of EEC from both large and small bowel to infection in vitro, using Chlamydia trachomatis infection as a model. METHODS: TWO HUMAN EEC LINES: LCC-18, derived from a neuroendocrine colonic tumour, and CNDT-2, derived from a small intestinal carcinoid, were infected using cultured C. trachomatis serovar LGV II strain 434 (ATCC VR-902B). Penicillin G was used to induce persistent infection. We used microarray analysis (Affymetrix GeneChip®) for studying changes in gene expression at different stages of infection. RESULTS: Twenty-four hours after active and persistent infection, 66 and 411 genes in LCC-18 and 68 and 170 genes in CNDT-2 cells, respectively showed mean expression ratios >2-fold compared to non-infected cells. These genes encoded factors regulating apoptosis, cell differentiation, transcription regulation, cytokine activity, amine biosynthesis and vesicular transport. We found significant differences in gene transcription levels between persistently infected and non-infected cells in 10 genes coding for different solute carrier transporters (SLC) and in 5 genes related to endocrine function (GABARAPL1, GRIP1, DRD2, SYT5 and SYT7). CONCLUSIONS: Infected EEC cells exhibit cell-type specific patterns related to vesicular transport, secretion and neurotransmitters. EEC play a pivotal role in regulation of gut motility and an impairment of enteroendocrine function can contribute to motility disorders.

Seroepidemiology of diphtheria and tetanus among children and young adults in Tajikistan: nationwide population-based survey, 2010.

BACKGROUND: Tajikistan had a major diphtheria outbreak (≈ 10,000 cases) in the 1990 s, which was controlled after nationwide immunization campaigns with diphtheria-tetanus toxoid in 1995 and 1996. Since 2000, only 52 diphtheria cases have been reported. However, in coverage surveys conducted in 2000 and 2005, diphtheria-tetanus-pertussis vaccine coverage was lower than administratively reported estimates raising concerns about potential immunity gaps. To further assess population immunity to diphtheria in Tajikistan, diphtheria antibody testing was included in a large-scale nationwide serosurvey for vaccine-preventable diseases conducted in connection with a poliomyelitis outbreak in 2010. In addition, the serosurvey provided an opportunity to assess population immunity to tetanus. METHODS: Residents of all regions of Tajikistan aged 1-24 years were included in the serosurvey implemented during September-October 2010. Participants were selected through stratified cluster sampling. Specimens were tested for diphtheria antibodies using a Vero cell neutralization assay and for tetanus antibodies using an anti-tetanus IgG ELISA. Antibody concentrations ≥ 0.1 IU/mL were considered seropositive. RESULTS: Overall, 51.4% (95% CI, 47.1%-55.6%) of participants were seropositive for diphtheria and 78.9% (95% CI, 74.7%-82.5%) were seropositive for tetanus. The lowest percentages of seropositivity for both diseases were observed among persons aged 10-19 years: diphtheria seropositivity was 37.1% (95% CI, 31.0%-43.7%) among 10-14 year-olds, and 35.3% (95% CI, 29.9%-41.1%) among 15-19 year-olds; tetanus seropositivity in respective age groups was 65.3% (95% CI, 58.4%-71.6%) and 70.1% (95% CI, 64.5%-75.2%). CONCLUSIONS: Population immunity for diphtheria in Tajikistan is low, particularly among 10-19 year-olds. Population immunity to tetanus is generally higher than for diphtheria, but is suboptimal among 10-19 year-olds. These findings highlight the need to improve routine immunization service delivery, and support a one-time supplementary immunization campaign with diphtheria-tetanus toxoid among birth cohorts aged 1-19 years in 2010 (3-21 years in 2012) to close immunity gaps and prevent diphtheria outbreaks.

Diphtheria in Europe: current problems and new challenges.

Diphtheria, caused by toxigenic strains of Corynebacterium diphtheriae, is an ancient disease with high incidence and mortality that has always been characterized by epidemic waves of occurrence. Whilst towards the beginning of the 1980s, many European countries were progressing towards the elimination of diphtheria, an epidemic re-emergence of diphtheria in the Russian Federation and the Newly Independent States of the former Soviet Union demonstrated a continuous threat of the disease into the 1990s. At present, the epidemic is under control and only sporadic cases are observed in Europe. However, the circulation of toxigenic strains is still observed in all parts of the world, posing a constant threat to the population with low levels of seroprotection. More recently, Corynebacterium ulcerans has been increasingly isolated as emerging zoonotic agent of diphtheria from companion animals such as cats or dogs, indicating the enduring threat of this thought-to-be controlled disease.

Molecular evolution of GII-4 Norovirus strains.

BACKGROUND: Human Noroviruses (NoV) are the major cause of acute nonbacterial gastroenteritis and the leading cause of outbreaks of gastroenteritis worldwide. Genotype II-4 (GII-4) NoV has been shown to spread rapidly and is the most commonly detected strain worldwide, particularly in association with outbreaks. Previously, we have shown that circulating GII-4 NoV strains exist as populations of selectively neutral variants, and that the emergence of epidemic GII-4 NoV strains correlated with mutations in at least two key sites (Sites A and B) within the P2 domain of the surface exposed major capsid protein (VP1). METHODOLOGY: We developed a rapid pyrosequencing method for screening of the two Sites A and B and a homology based modelling system was used to predict the effects of amino acid substitutions at these sites on the antigenic properties of the virus (defined as surface motif types). PRINCIPLE FINDING/CONCLUSION: Here, we describe the characterisation of amino acid diversity at Sites A and B for 1062 GII-4 NoV strains from clinical specimen associated with outbreak of gastroenteritis (2000-2011) and 250 GII-4 NoV sequences from Genbank. Our data identified a high diversity of different Site A and B site combinations at amino acid level and amino acid diversity was higher at Site B than Site A. Site A motifs could be grouped into 3 clusters based on similar surface motif types. We predict that Site A is a major epitope on the virus surface, responsible for defining the antigenic profile, and a more subtle role for Site B, maintaining minor antigenic variation within the virus population.

In vivo transcript profiling of Candida albicans identifies a gene essential for interepithelial dissemination.

Candida albicans is the most common oral fungal pathogen of humans, but the mechanisms by which C. albicans invades and persists within mucosal epithelium are not clear. To understand oral pathogenesis, we characterized the cellular and molecular mechanisms of epithelial-fungus interactions using reconstituted human oral epithelium (RHE). We observed that hyphal formation facilitates epithelial invasion via both active (physical penetration) and passive (induced endocytosis) processes. Genome wide transcript profiling of C. albicans experimental RHE infection was compared with that from 11 patient samples with pseudomembranous candidiasis to identify genes associated with disease development in vivo. Expression profiles reflected the morphological switch and an adaptive response to neutral pH, non-glucose carbon sources and nitrosative stress. We identified several novel infection-associated genes with unknown function. One gene, upregulated in both RHE infection and patients, named EED1, was essential for maintenance of hyphal elongation. Mutants lacking EED1 showed transient cell elongation on epithelial tissue, which enabled only superficial invasion of epithelial cells. Once inside an epithelial cell, Deltaeed1 cells could proliferate as yeasts or pseudohyphae but remained trapped intracellularly. Our results suggest that the adaptive response and morphology of C. albicans play specific roles for host-fungal interactions during mucosal infections.

PGA4, a GAS homologue from Candida albicans, is up-regulated early in infection processes.

Transglucosidases play a significant role in fungal cell wall biosynthesis. We identified three as yet undescribed genes encoding beta-glucan transglucosidases, homologues of the pH-regulated PHR1 and PHR2, in the genome of the pathogenic yeast Candida albicans. Transcript levels of the gene PGA4 encoding a putative GPI-anchored protein were elevated in C. albicans wild-type cells during infection of reconstituted human epithelial and mouse liver tissue, and transiently increased after induction of hyphal formation with serum. The serum-specific increase in PGA4 transcript was found to be dependent on the transcription factors Ras1p, Cyr1p, and Tec1p. The remaining C. albicans Phr homologues, PHR3 and PGA5, showed low expression levels. Unlike PHR1 and PHR2, the expression of PHR3, PGA4, and PGA5 was not dependent on the pH of the growth medium. Neither PHR3 deletion nor PGA4 disruption resulted in a distinct growth or morphology phenotype. A PGA4 disruption strain was found to have wild-type capacity of infecting reconstituted oral epithelial tissue. Our data suggest that PGA4, and potentially PHR3 and PGA5, are expressed under distinct conditions, which differ from those of PHR1 and PHR2.

Models of oral and vaginal candidiasis based on in vitro reconstituted human epithelia.

This protocol describes the setup, maintenance and characteristics of models of epithelial Candida infections based on well-established three-dimensional organotypic tissues of human oral and vaginal mucosa. Infection experiments are highly reproducible and can be used for the direct analysis of pathogen-epithelial cell interactions. This allows detailed investigations of Candida albicans wild type or mutant strain interaction with epithelial tissue or the evaluation of the host immune response using histological, biochemical and molecular methods. As such, the models can be utilized as a tool to investigate cellular interactions or protein and gene expression that are not complicated by non-epithelial factors. To study the impact of innate immunity or the antifungal activity of natural and non-natural compounds, the mucosal infection models can be supplemented with immune cells, antimicrobial agents or probiotic bacteria. The model requires at least 3 days to be established and can be maintained thereafter for 2-4 days.