Candida albicans possess a highly versatile and dynamic high‐affinity iron transport system important for its commensal‐pathogenic lifestyle

Iron is an essential nutrient for nearly all organisms, but iron overdose is toxic. The human commensal‐pathogenic fungus Candida albicans traverses host niches with markedly different iron availability. During systemic infection, C. albicans must activate the high‐affinity iron permease Ftr1 to acquire iron sequestered by the host's iron‐withholding defense and suppresses iron uptake while residing in the iron‐rich gut to avoid toxicity. Ftr1 associates with a ferroxidase to form an iron transporter. C. albicans contains four permeases and five ferroxidase homologs, suggesting 20 possible subunit combinations. Here, we investigated the iron‐dependent expression, cellular localization and interacting partners of all permeases and ferroxidases and the significance of each subunit for gastrointestinal colonization and systemic infection in mice. We uncovered three distinct patterns of iron‐dependent expression and highly flexible ferroxidase‐permease partnerships, which underlie a dynamic iron transport system that can be deftly tuned according to iron availability. We found functional differentiation as well as redundancy among the ferroxidases and permeases during both gastrointestinal colonization and bloodstream infection. We propose that C. albicans possesses a sophisticated iron acquisition and utilization system befitting its commensal‐pathogenic lifestyle. Our findings reveal new possibilities for medical intervention of C. albicans infection.     

Candida albicans colonization and dissemination from the murine gastrointestinal tract: the influence of morphology and Th17 immunity

The ability of Candida albicans to cause disease is associated with its capacity to undergo morphological transition between yeast and filamentous forms, but the role of morphology in colonization and dissemination from the gastrointestinal (GI) tract remains poorly defined. To explore this, we made use of wild‐type and morphological mutants of C. albicans in an established model of GI tract colonization, induced following antibiotic treatment of mice. Our data reveal that GI tract colonization favours the yeast form of C. albicans, that there is constitutive low level systemic dissemination in colonized mice that occurs irrespective of fungal morphology, and that colonization is not controlled by Th17 immunity in otherwise immunocompetent animals. These data provide new insights into the mechanisms of pathogenesis and commensalism of C. albicans, and have implications for our understanding of human disease.     

Murine Models of Candida Gastrointestinal Colonization and Dissemination

Ninety-five percent of infectious agents enter through exposed mucosal surfaces, such as the respiratory and gastrointestinal (GI) tracts. The human GI tract is colonized with trillions of commensal microbes, including numerous Candida spp. Some commensal microbes in the GI tract can cause serious human infections under specific circumstances, typically involving changes in the gut environment and/or host immune conditions. Therefore, utilizing animal models of fungal GI colonization and dissemination can lead to significant insights into the complex pathophysiology of transformation from a commensal organism to a pathogen and host-pathogen interactions. This paper will review the methodologic approaches used for modeling GI colonization versus dissemination, the insights learned from these models, and finally, possible future directions using these animal modeling systems.     

Enemies and brothers in arms: Candida albicans and gram‐positive bacteria

Candida albicans is an important human opportunistic fungal pathogen which is frequently found as part of the normal human microbiota. It is well accepted that the fungus interacts with other components of the resident microbiota and that this impacts the commensal or pathogenic outcome of C. albicans colonization. Different types of interactions, including synergism or antagonism, contribute to a complex balance between the multitude of different species. Mixed biofilms of C. albicans and streptococci are a well‐studied example of a mutualistic interaction often potentiating the virulence of the individual members. In contrast, other bacteria like lactobacilli are known to antagonize C. albicans, and research has just started elucidating the mechanisms behind these interactions. This scenario is even more complicated by a third player, the host. This review focuses on interactions between C. albicans and gram‐positive bacteria whose investigation will without doubt ultimately help understanding C. albicans infections.     

Gastrointestinal Colonization of Fungi

The human gastrointestinal tract is colonized with trillions of commensal microbes, and disturbances in the equilibrium of the gut microbiota have now been shown to be associated with a number of human diseases. Fungi, particularly Candida spp., are normal, harmless residents of the human gut, but in certain instances can cause invasive infections and inflammatory disorders. This paper will review the fungal diversity in the human gut, host and fungal factors that regulate GI colonization, and how these factors play into the pathogenesis of human disease.     

Candida albicans Niche Specialization: Features That Distinguish Biofilm Cells from Commensal Cells

The fungus Candida albicans is a frequent commensal colonizer of the human gastrointestinal (GI) tract, but is also an opportunistic pathogen. This review explores features that distinguish the colonizing and pathogenic forms of C. albicans. Candida albicans in a biofilm is used as an example of a pathogenic form of the organism, because biofilms are a common feature of device-associated C. albicans infections. Biofilms (complex, sessile communities of cells) have been the subject of several large-scale gene expression studies. Biofilms and commensal C. albicans colonizing the murine GI tract show a variety of differentially expressed genes. Cell surface proteins encoded by these differentially expressed genes are especially attractive as targets for new clinical prevention, diagnosis, or treatment tools that are specific for C. albicans in its pathogenic biofilm state.     

Intestinal Colonization by Candida albicans Alters Inflammatory Responses in Bruton's Tyrosine Kinase-Deficient Mice

The commensal yeast Candida albicans is part of the human intestinal microflora and is considered a “pathobiont”, a resident microbe with pathogenic potential yet harmless under normal conditions. The aim of this study was to investigate the effect of C. albicans on inflammation of the intestinal tract and the role of Bruton''s tyrosine kinase (Btk). Btk is an enzyme that modulates downstream signaling of multiple receptors involved in innate and adaptive immunity, including the major anti-fungal receptor Dectin-1. Colitis was induced in wild type and Btk-/- mice by treatment with dextran sodium sulfate (DSS) and the gastrointestinal tract of selected treatment groups were then colonized with C. albicans. Colonization by C. albicans neither dampened nor exacerbated inflammation in wild type mice, but colon length and spleen weight were improved in Btk-deficient mice colonized with C. albicans. Neutrophil infiltration was comparable between wild type and Btk-/- mice, but the knockout mice displayed severely reduced numbers of macrophages in the colon during both DSS and DSS/Candida treatment. Smaller numbers and reduced responsiveness of Btk-/- macrophages might partially explain the improved colon length of Btk-/- mice as a result of Candida colonization. Surprisingly, DSS/Candida-treated Btk-/- animals had higher levels of certain pro-inflammatory cytokines and levels of the anti-inflammatory cytokine TGF-β were reduced compared to wild type. A clustering and correlation analysis showed that for wild type animals, spleen TGF-β and colon IL-10 and for Btk-/- spleen and colon levels of IL-17A best correlated with the inflammatory parameters. We conclude that in Btk-/- immunocompromised animals, colonization of the gastrointestinal tract by the commensal yeast C. albicans alters inflammatory symptoms associated with colitis.     

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.Download video file.^{(143M, mp4)}     

The HOG Pathway Is Critical for the Colonization of the Mouse Gastrointestinal Tract by Candida albicans

The opportunistic pathogen Candida albicans is a frequent inhabitant of the human gastrointestinal tract where it usually behaves as a harmless commensal. In this particular niche, it needs to adapt to the different micro environments that challenge its survival within the host. In order to determine those factors involved in gut adaptation, we have used a gastrointestinal model of colonization in mouse to trace the behaviour of fungal cells. We have developed a genetic labelling system based on the complementary spectral properties of the fluorescent proteins GFP and a new C. albicans codon-adapted RFP (dTOM2) that allow a precise quantification of the fungal population in the gut via standard in vitro cultures or flow cytometry. This methodology has allowed us to determine the role of the three MAP kinase pathways of C. albicans (mediated by the MAPK Mkc1, Cek1 or Hog1) in mouse gut colonization via competitive assays with MAPK pathway mutants and their isogenic wild type strain. This approach reveals the signalling through HOG pathway as a critical factor influencing the establishment of C. albicans in the mouse gut. Less pronounced effects for mkc1 or cek1 mutants were found, only evident after 2–3 weeks of colonization. We have also seen that hog1 mutants is defective in adhesion to the gut mucosa and sensitive to bile salts. Finally, we have developed a genetic strategy for the in vivo excision (tetracycline-dependent) of any specific gene during the course of colonization in this particular niche, allowing the analysis of its role during gut colonization.     

Commensal microbiota influence systemic autoimmune responses

Antinuclear antibodies are a hallmark feature of generalized autoimmune diseases, including systemic lupus erythematosus and systemic sclerosis. However, the processes underlying the loss of tolerance against nuclear self‐constituents remain largely unresolved. Using mice deficient in lymphotoxin and Hox11, we report that approximately 25% of mice lacking secondary lymphoid organs spontaneously develop specific antinuclear antibodies. Interestingly, we find this phenotype is not caused by a defect in central tolerance. Rather, cell‐specific deletion and in vivo lymphotoxin blockade link these systemic autoimmune responses to the formation of gut‐associated lymphoid tissue in the neonatal period of life. We further demonstrate antinuclear antibody production is influenced by the presence of commensal gut flora, in particular increased colonization with segmented filamentous bacteria, and IL‐17 receptor signaling. Together, these data indicate that neonatal colonization of gut microbiota influences generalized autoimmunity in adult life.     

Microbiome precision editing: Using PEG as a selective fermentation initiator against methicillin‐resistant Staphylococcus aureus

Recent creation of a Unified Microbiome Initiative (UMI) has the aim of understanding how microbes interact with each other and with us. When pathogenic Staphylococcus aureus infects the skin, the interplay between S. aureus and skin commensal bacteria occurs. Our previous data revealed that skin commensal bacteria can mediate fermentation against the growth of USA300, a community‐acquired methicillin‐resistant S. aureus MRSA. By using a fermentation process with solid media on a small scale, we define poly(ethylene glycol) dimethacrylate (PEG‐DMA) as a selective fermentation initiator which can specifically intensify the probiotic ability of skin commensal Staphylococcus epidermidis bacteria. At least five short‐chain fatty acids including acetic, butyric and propionic acids with anti‐USA300 activities are produced by PEG‐DMA fermentation of S. epidermidis. Furthermore, the S. epidermidis‐laden PEG‐DMA hydrogels effectively decolonized USA300 in skin wounds in mice. The PEG‐DMA and its derivatives may become novel biomaterials to specifically tailor the human skin microbiome against invading pathogens.     

Structural basis for adaptation of lactobacilli to gastrointestinal mucus

The mucus layer covering the gastrointestinal (GI) epithelium is critical in selecting and maintaining homeostatic interactions with our gut bacteria. However, the underpinning mechanisms of these interactions are not understood. Here, we provide structural and functional insights into the canonical mucus‐binding protein (MUB), a multi‐repeat cell‐surface adhesin found in Lactobacillus inhabitants of the GI tract. X‐ray crystallography together with small‐angle X‐ray scattering demonstrated a ‘beads on a string’ arrangement of repeats, generating 174 nm long protein fibrils, as shown by atomic force microscopy. Each repeat consists of tandemly arranged Ig‐ and mucin‐binding protein (MucBP) modules. The binding of full‐length MUB was confined to mucus via multiple interactions involving terminal sialylated mucin glycans. While individual MUB domains showed structural similarity to fimbrial proteins from Gram‐positive pathogens, the particular organization of MUB provides a structural explanation for the mechanisms in which lactobacilli have adapted to their host niche by maximizing interactions with the mucus receptors, potentiating the retention of bacteria within the mucus layer. Together, this study reveals functional and structural features which may affect tropism of microbes across mucus and along the GI tract, providing unique insights into the mechanisms adopted by commensals and probiotics to adapt to the mucosal environment.     

Modulation of human endogenous retroviruses –H, -W and -K transcription by microbes

The human endogenous retroviruses (HERVs) are endogenous retroviruses that are inserted into the germ cell DNA of humans over 30 million years ago. Using real-time RT-PCR we describe HERV modulation by commensal microbes in the human gut.Infants, exclusively or predominant breast milk feeding, less than 12 weeks of age, during bacteria gut colonization, were assessed for eligibility.Our data demonstrate that the colonization with commensal microbes, in particular, Bifidobacterium spp., of the gut causes modulation of HERVs.     

ACC deaminase‐containing plant growth‐promoting rhizobacteria protect Papaver somniferum from downy mildew

Aims

Analysis of the stability and safety of Sulfolobus monocaudavirus 1 (SMV1) during passage through the mammalian GI tract.

Methods and Results

A major challenge of using nano‐vectors to target gut microbiome is their survival during passage through the extremely acidic and proteolytic environment of the mammalian GI tract. Here, we investigated the thermo‐acidophilic archaeal virus SMV1 as a candidate therapeutic nano‐vector for the distal mammalian GI tract microbiome. We investigated the anatomical distribution, vector stability and immunogenicity of this virus following oral ingestion in mice and compared these traits to the more classically used Inovirus vector M13KE. We found that SMV1 particles were highly stable under both simulated GI tract conditions (in vitro) and in mice (in vivo). Moreover, SMV1 could not be detected in tissues outside the GI tract and it elicited a nearly undetectable inflammatory response. Finally, we used human intestinal organoids (HIOs) to show that labelled SMV1 did not invade or otherwise perturb the human GI tract epithelium.

Conclusion

Sulfolobus monocaudavirus 1 appeared stable and safe during passage though the mammalian GI tract.

Significance and Impact of the Study

This is the first study evaluating an archaeal virus as a potential therapeutic nanoparticle delivery system and it opens new possibilities for future development of novel nanoplatforms.     

Glutamate decarboxylase‐dependent acid resistance in orally acquired bacteria: function,distribution and biomedical implications of the gadBC operon

For successful colonization of the mammalian host, orally acquired bacteria must overcome the extreme acidic stress (pH < 2.5) encountered during transit through the host stomach. The glutamate‐dependent acid resistance (GDAR) system is by far the most potent acid resistance system in commensal and pathogenic Escherichia coli, Shigella flexneri, Listeria monocytogenes and Lactococcus lactis. GDAR requires the activity of glutamate decarboxylase (GadB), an intracellular PLP‐dependent enzyme which performs a proton‐consuming decarboxylation reaction, and of the cognate antiporter (GadC), which performs the glutamate_in/γ‐aminobutyrate_out (GABA) electrogenic antiport. Herein we review recent findings on the structural determinants responsible for pH‐dependent intracellular activation of E. coli GadB and GadC. A survey of genomes of bacteria (pathogenic and non‐pathogenic), having in common the ability to colonize or to transit through the host gut, shows that the gadB and gadC genes frequently lie next or near each other. This gene arrangement is likely to be important to ensure timely co‐regulation of the decarboxylase and the antiporter. Besides the involvement in acid resistance, GABA production and release were found to occur at very high levels in lactic acid bacteria originally isolated from traditionally fermented foods, supporting the evidence that GABA‐enriched foods possess health‐promoting properties.     

Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox‐mediated generation of reactive oxygen species

The resident prokaryotic microbiota of the metazoan gut elicits profound effects on the growth and development of the intestine. However, the molecular mechanisms of symbiotic prokaryotic–eukaryotic cross‐talk in the gut are largely unknown. It is increasingly recognized that physiologically generated reactive oxygen species (ROS) function as signalling secondary messengers that influence cellular proliferation and differentiation in a variety of biological systems. Here, we report that commensal bacteria, particularly members of the genus Lactobacillus, can stimulate NADPH oxidase 1 (Nox1)‐dependent ROS generation and consequent cellular proliferation in intestinal stem cells upon initial ingestion into the murine or Drosophila intestine. Our data identify and highlight a highly conserved mechanism that symbiotic microorganisms utilize in eukaryotic growth and development. Additionally, the work suggests that specific redox‐mediated functions may be assigned to specific bacterial taxa and may contribute to the identification of microbes with probiotic potential.     

Like Will to Like: Abundances of Closely Related Species Can Predict Susceptibility to Intestinal Colonization by Pathogenic and Commensal Bacteria

The intestinal ecosystem is formed by a complex, yet highly characteristic microbial community. The parameters defining whether this community permits invasion of a new bacterial species are unclear. In particular, inhibition of enteropathogen infection by the gut microbiota ( = colonization resistance) is poorly understood. To analyze the mechanisms of microbiota-mediated protection from Salmonella enterica induced enterocolitis, we used a mouse infection model and large scale high-throughput pyrosequencing. In contrast to conventional mice (CON), mice with a gut microbiota of low complexity (LCM) were highly susceptible to S. enterica induced colonization and enterocolitis. Colonization resistance was partially restored in LCM-animals by co-housing with conventional mice for 21 days (LCM^con21). 16S rRNA sequence analysis comparing LCM, LCM^con21 and CON gut microbiota revealed that gut microbiota complexity increased upon conventionalization and correlated with increased resistance to S. enterica infection. Comparative microbiota analysis of mice with varying degrees of colonization resistance allowed us to identify intestinal ecosystem characteristics associated with susceptibility to S. enterica infection. Moreover, this system enabled us to gain further insights into the general principles of gut ecosystem invasion by non-pathogenic, commensal bacteria. Mice harboring high commensal E. coli densities were more susceptible to S. enterica induced gut inflammation. Similarly, mice with high titers of Lactobacilli were more efficiently colonized by a commensal Lactobacillus reuteri ^RR strain after oral inoculation. Upon examination of 16S rRNA sequence data from 9 CON mice we found that closely related phylotypes generally display significantly correlated abundances (co-occurrence), more so than distantly related phylotypes. Thus, in essence, the presence of closely related species can increase the chance of invasion of newly incoming species into the gut ecosystem. We provide evidence that this principle might be of general validity for invasion of bacteria in preformed gut ecosystems. This might be of relevance for human enteropathogen infections as well as therapeutic use of probiotic commensal bacteria.