Host carbon sources modulate cell wall architecture, drug resistance and virulence in a fungal pathogen

The survival of all microbes depends upon their ability to respond to environmental challenges. To establish infection, pathogens such as Candida albicans must mount effective stress responses to counter host defences while adapting to dynamic changes in nutrient status within host niches. Studies of C. albicans stress adaptation have generally been performed on glucose‐grown cells, leaving the effects of alternative carbon sources upon stress resistance largely unexplored. We have shown that growth on alternative carbon sources, such as lactate, strongly influence the resistance of C. albicans to antifungal drugs, osmotic and cell wall stresses. Similar trends were observed in clinical isolates and other pathogenic Candida species. The increased stress resistance of C. albicans was not dependent on key stress (Hog1) and cell integrity (Mkc1) signalling pathways. Instead, increased stress resistance was promoted by major changes in the architecture and biophysical properties of the cell wall. Glucose‐ and lactate‐grown cells displayed significant differences in cell wall mass, ultrastructure, elasticity and adhesion. Changes in carbon source also altered the virulence of C. albicans in models of systemic candidiasis and vaginitis, confirming the importance of alternative carbon sources within host niches during C. albicans infections.     

Carbon source‐induced reprogramming of the cell wall proteome and secretome modulates the adherence and drug resistance of the fungal pathogen Candida albicans

The major fungal pathogen Candida albicans can occupy diverse microenvironments in its human host. During colonization of the gastrointestinal or urogenital tracts, mucosal surfaces, bloodstream, and internal organs, C. albicans thrives in niches that differ with respect to available nutrients and local environmental stresses. Although most studies are performed on glucose‐grown cells, changes in carbon source dramatically affect cell wall architecture, stress responses, and drug resistance. We show that growth on the physiologically relevant carboxylic acid, lactate, has a significant impact on the C. albicans cell wall proteome and secretome. The regulation of cell wall structural proteins (e.g. Cht1, Phr1, Phr2, Pir1) correlated with extensive cell wall remodeling in lactate‐grown cells and with their increased resistance to stresses and antifungal drugs, compared with glucose‐grown cells. Moreover, changes in other proteins (e.g. Als2, Gca1, Phr1, Sap9) correlated with the increased adherence and biofilm formation of lactate‐grown cells. We identified mating and pheromone‐regulated proteins that were exclusive to lactate‐grown cells (e.g. Op4, Pga31, Pry1, Scw4, Yps7) as well as mucosa‐specific and other niche‐specific factors such as Lip4, Pga4, Plb5, and Sap7. The analysis of the corresponding null mutants confirmed that many of these proteins contribute to C. albicans adherence, stress, and antifungal drug resistance. Therefore, the cell wall proteome and secretome display considerable plasticity in response to carbon source. This plasticity influences important fitness and virulence attributes known to modulate the behavior of C. albicans in different host microenvironments during infection.     

The carboxylic acid transporters Jen1 and Jen2 affect the architecture and fluconazole susceptibility of Candida albicans biofilm in the presence of lactate

Candida albicans has the ability to adapt to different host niches, often glucose-limited but rich in alternative carbon sources. In these glucose-poor microenvironments, this pathogen expresses JEN1 and JEN2 genes, encoding carboxylate transporters, which are important in the early stages of infection. This work investigated how host microenvironments, in particular acidic containing lactic acid, affect C. albicans biofilm formation and antifungal drug resistance. Multiple components of the extracellular matrix were also analysed, including their impact on antifungal drug resistance, and the involvement of both Jen1 and Jen2 in this process. The results show that growth on lactate affects biofilm formation, morphology and susceptibility to fluconazole and that both Jen1 and Jen2 might play a role in these processes. These results support the view that the adaptation of Candida cells to the carbon source present in the host niches affects their pathogenicity.     

Functional specialization and differential regulation of short‐chain carboxylic acid transporters in the pathogen Candida albicans

The major fungal pathogen Candida albicans has the metabolic flexibility to assimilate a wide range of nutrients in its human host. Previous studies have suggested that C. albicans can encounter glucose‐poor microenvironments during infection and that the ability to use alternative non‐fermentable carbon sources contributes to its virulence. JEN1 encodes a monocarboxylate transporter in C. albicans and we show that its paralogue, JEN2, encodes a novel dicarboxylate plasma membrane transporter, subjected to glucose repression. A strain deleted in both genes lost the ability to transport lactic, malic and succinic acids by a mediated mechanism and it displayed a growth defect on these substrates. Although no significant morphogenetic or virulence defects were found in the double mutant strain, both JEN1 and JEN2 were strongly induced during infection. Jen1‐GFP (green fluorescent protein) and Jen2‐GFP were upregulated following the phagocytosis of C. albicans cells by neutrophils and macrophages, displaying similar behaviour to an Icl1‐GFP fusion. In the murine model of systemic candidiasis approximately 20–25% of C. albicans cells infecting the kidney expressed Jen1‐GFP and Jen2‐GFP. Our data suggest that Jen1 and Jen2 are expressed in glucose‐poor niches within the host, and that these short‐chain carboxylic acid transporters may be important in the early stages of infection.     

Stress magnitude matters: different intensities of pulsed water stress produce non‐monotonic resistance responses of host plants to insect herbivores

Abstract 1. Water stress may increase or reduce the suitability of plants for herbivores. The recently proposed ‘pulsed stress hypothesis’ suggests consideration of stress phenology (pulsed vs. continuous stress) to explain these conflicting effects of plant water stress on herbivore performance. 2. This hypothesis was tested for the effect of differing stress intensity on performance and preference of insect herbivores belonging to different feeding guilds, namely leaf‐chewing insects (Spodoptera littoralis caterpillars) and phloem‐feeding insects (Aphis pomi aphids), on apple plants (Malus domestica). The plants were non‐stressed or exposed to a low or high intensity of pulsed water stress. 3. Plant responses to the different stress levels were generally monotonic. Growth, stomatal conductance (g_s), leaf water, and old‐leaf nitrogen concentration decreased, whereas young‐leaf nitrogen concentration and leaf mass per area (LMA) increased with increasing stress intensity. The stable isotope composition of foliar carbon (δ¹³C) responded non‐monotonically to the drought treatments. The δ¹³C values were highest in low‐stress plants, intermediate in high‐stress plants, and lowest in non‐stressed plants. 4. The preference and performance responses of the caterpillars were also non‐monotonic. Non‐stressed plants were intermediately, low‐stress plants least, and high‐stress plants most attractive or suitable. Aphid population growth was highest on non‐stressed plants and lowest on low‐stress plants. 5. The results highlight the importance of water stress intensity for the outcome of interactions between herbivores and drought‐affected plants. They show that pulsed water stress may enhance or reduce insect herbivore performance and plant resistance, depending on stress intensity.     

Protective effect of fungal extracellular vesicles against murine candidiasis

Extracellular vesicles (EVs) are lipid bilayered compartments released by virtually all living cells, including fungi. Among the diverse molecules carried by fungal EVs, a number of immunogens, virulence factors and regulators have been characterised. Within EVs, these components could potentially impact disease outcomes by interacting with the host. From this perspective, we previously demonstrated that EVs from Candida albicans could be taken up by and activate macrophages and dendritic cells to produce cytokines and express costimulatory molecules. Moreover, pre‐treatment of Galleria mellonella larvae with fungal EVs protected the insects against a subsequent lethal infection with C. albicans yeasts. These data indicate that C. albicans EVs are multi‐antigenic compartments that activate the innate immune system and could be exploited as vaccine formulations. Here, we investigated whether immunisation with C. albicans EVs induces a protective effect against murine candidiasis in immunosuppressed mice. Total and fungal antigen‐specific serum IgG antibodies increased by 21 days after immunisation, confirming the efficacy of the protocol. Vaccination decreased fungal burden in the liver, spleen and kidney of mice challenged with C. albicans. Splenic levels of cytokines indicated a lower inflammatory response in mice immunised with EVs when compared with EVs + Freund's adjuvant (ADJ). Higher levels of IL‐12p70, TNFα and IFNγ were detected in mice vaccinated with EVs + ADJ, while IL‐12p70, TGFβ, IL‐4 and IL‐10 were increased when no adjuvants were added. Full protection of lethally challenged mice was observed when EVs were administered, regardless the presence of adjuvant. Physical properties of the EVs were also investigated and EVs produced by C. albicans were relatively stable after storage at 4, ?20 or ?80°C, keeping their ability to activate dendritic cells and to protect G. mellonella against a lethal candidiasis. Our data suggest that fungal EVs could be a safe source of antigens to be exploited in vaccine formulations.     

Towards non‐invasive monitoring of pathogen–host interactions during Candida albicans biofilm formation using in vivo bioluminescence

Candida albicans is a major human fungal pathogen causing mucosal and deep tissue infections of which the majority is associated with biofilm formation on medical implants. Biofilms have a huge impact on public health, as fungal biofilms are highly resistant against most antimycotics. Animal models of biofilm formation are indispensable for improving our understanding of biofilm development inside the host, their antifungal resistance and their interaction with the host immune defence system. In currently used models, evaluation of biofilm development or the efficacy of antifungal treatment is limited to ex vivo analyses, requiring host sacrifice, which excludes longitudinal monitoring of dynamic processes during biofilm formation in the live host. In this study, we have demonstrated for the first time that non‐invasive, dynamic imaging and quantification of in vitro and in vivo C. albicans biofilm formation including morphogenesis from the yeast to hyphae state is feasible by using growth‐phase dependent bioluminescent C. albicans strains in a subcutaneous catheter model in rodents. We have shown the defect in biofilm formation of a bioluminescent bcr1 mutant strain. This approach has immediate applications for the screening and validation ofantimycotics under in vivo conditions, for studying host–biofilm interactions in different transgenic mouse models and for testing the virulence of luminescent C. albicans mutants, hereby contributing to a better understanding of the pathogenesis of biofilm‐associated yeast infections.     

Virulence and pathogenicity of a Candida albicans mutant with reduced filamentation

Deletion of DNA polymerase eta (Rad30/Polη) in pathogenic yeast Candida albicans is known to reduce filamentation induced by serum, ultraviolet, and cisplatin. Because nonfilamentous C. albicans is widely accepted as avirulent form, here we explored the virulence and pathogenicity of a rad30Δ strain of C. albicans in cell‐based and animal systems. Flow cytometry of cocultured fungal and differentiated macrophage cells revealed that comparatively higher percentage of macrophages was associated with the wild‐type than rad30Δ cells. In contrast, higher number of Polη‐deficient C. albicans adhered per macrophage membrane. Imaging flow cytometry showed that the wild‐type C. albicans developed hyphae after phagocytosis that caused necrotic death of macrophages to evade their clearance. Conversely, phagosomes kill the fungal cells as estimated by increased metacaspase activity in wild‐type C. albicans. Despite the morphological differences, both wild‐type and rad30? C. albicans were virulent with a varying degree of pathogenicity in mice models. Notably, mice with Th1 immunity were comparatively less susceptible to systemic fungal infection than Th2 type. Thus, our study clearly suggests that the modes of interaction of morphologically different C. albicans strains with the host immune cells are diverged, and host genetic background and several other attributing factors of the fungus could additionally determine their virulence.     

Regulation of Rfa2 phosphorylation in response to genotoxic stress in Candida albicans

Successful pathogens must be able to swiftly respond to and repair DNA damages inflicted by the host defence. The replication protein A (RPA) complex plays multiple roles in DNA damage response and is regulated by phosphorylation. However, the regulators of RPA phosphorylation remain unclear. Here, we investigated Rfa2 phosphorylation in the pathogenic fungus Candida albicans. Rfa2, a RFA subunit, is phosphorylated when DNA replication is inhibited by hydroxyurea and dephosphorylated during the recovery. By screening a phosphatase mutant library, we found that Pph3 associates with different regulatory subunits to differentially control Rfa2 dephosphorylation in stressed and unstressed cells. Site‐directed mutagenesis revealed T11, S18, S29, and S30 being critical for Rfa2 phosphorylation in response to genotoxic insult. We obtained evidence that the genome integrity checkpoint kinase Mec1 and the cyclin‐dependent kinase Clb2–Cdc28 mediate Rfa2 phosphorylation. Although cells expressing either a phosphomimetic or a non‐phosphorylatable version of Rfa2 had defects, the latter exhibited greater sensitivity to genotoxic challenge, failure to repair DNA damages and to deactivate Rad53‐mediated checkpoint pathways in a dosage‐dependent manner. These mutants were also less virulent in mice. Our results provide important new insights into the regulatory mechanism and biological significance of Rfa2 phosphorylation in C. albicans.     

Two respiratory enzyme systems in Campylobacter jejuni NCTC 11168 contribute to growth on l‐lactate

Campylobacter jejuni, a major food‐borne intestinal pathogen, preferentially utilizes a few specific amino acids and some organic acids such as pyruvate and l ‐ and d ‐lactate as carbon sources, which may be important for growth in the avian and mammalian gut. Here, we identify the enzymatic basis for C. jejuni growth on l ‐lactate. Despite the presence of an annotated gene for a fermentative lactate dehydrogenase (cj1167), no evidence for lactate excretion could be obtained in C. jejuni NCTC 11168, and inactivation of the cj1167 gene did not affect growth on lactate as carbon source. Instead, l ‐lactate utilization in C. jejuni NCTC 11168 was found to proceed via two novel NAD‐independent l ‐LDHs; a non‐flavin iron–sulfur containing three subunit membrane‐associated enzyme (Cj0075c‐73c), and a flavin and iron–sulfur containing membrane‐associated oxidoreductase (Cj1585c). Both enzymes contribute to growth on l ‐lactate, as single mutants in each system grew as well as wild‐type on this substrate, while a cj0075c cj1585c double mutant showed no l ‐lactate oxidase activity and did not utilize or grow on l ‐lactate; d ‐lactate‐dependent growth was unaffected. Orthologues of Cj0075c‐73c (LldEFG/LutABC) and Cj1585c (Dld‐II) were recently shown to represent two novel families of l ‐ and d ‐lactate oxidases; this is the first report of a bacterium where both enzymes are involved in l ‐lactate utilization only. The cj0075c‐73c genes are located directly downstream of a putative lactate transporter gene (cj0076c, lctP), which was also shown to be specific for l ‐lactate. The avian and mammalian gut environment contains dense populations of obligate anaerobes that excrete lactate; our data indicate that C. jejuni is well equipped to use l ‐ and d ‐lactate as both electron‐donor and carbon source.