Mapping the mutual transcriptional responses during Candida albicans and human macrophage interactions by dual RNA-sequencing.

Candida albicans is the leading human fungal pathogen that can cause mucosal and systemic fungal infections. Host phagocytes are the primary immune defense against invading fungal pathogens including C. albicans. To better understand the host-pathogen interaction between C. albicans and host phagocytes, we utilized a human macrophage model of THP-1 macrophages and examined the mutual transcriptomic response of C. albicans and host macrophages by dual RNA-sequencing. Both C. albicans and macrophages displayed marked changes in their transcriptional profiles post 2 h coincubation. We show that C. albicans responds to human macrophages differently than its known response to murine macrophages. C. albicans displays upregulation of its translational machinery and downregulation of glyoxylate and tricarboxylic acid (TCA) cycle upon macrophage phagocytosis. C. albicans triggered strong induction of genes associated with cell surface-mediated signaling and proinflammatory response in THP-1 macrophages. Finally, our data reveal that IL-1ß and TNF signaling are central in mounting a proinflammatory response against C. albicans via MAP kinase, and chemokines and cytokines mediated signaling. Overall, current work uncovers the mutual responses of C. albicans and human macrophages towards each other presenting a better understanding of their interaction during C. albicans infections.

Genome Sequencing and Functional Characterization of Xanthomonas cucurbitae, the Causal Agent of Bacterial Spot Disease of Cucurbits.

Bacterial leaf spot disease caused by Xanthomonas cucurbitae has severely affected the pumpkin industries in the Midwestern region of United States, with the bacteria mainly infecting pumpkin leaves and fruits, and leading to significant yield losses. In this study, we utilized genomics and genetics approaches to elucidate X. cucurbitae molecular mechanisms of pathogenesis during interaction with its host. We generated the first reference-quality whole-genome sequence of the X. cucurbitae type isolate and compared with other Xanthomonas species, X. cucurbitae has a smaller genome size with fewer virulence-related genes. RNA-seq analysis of X. cucurbitae under plant-mimicking media conditions showed altered transcriptional responses, with upregulation of virulence genes and downregulation of cellular homeostasis genes. Additionally, characterization of key virulence genes using gene deletion methods revealed that both type II enzymes and type III effectors are necessary for X. cucurbitae to cause infection in the pumpkin host.

Cell-cell signalling promotes ferric iron uptake in Xanthomonas oryzae pv. oryzicola that contribute to its virulence and growth inside rice.

Cell-cell communication mediated by diffusible signal factor (DSF) plays an important role in virulence of several Xanthomonas group of plant pathogens. In the bacterial pathogen of rice, Xanthomonas oryzae pv. oryzicola, DSF is required for virulence and in planta growth. In order to understand the role of DSF in promoting in planta growth and virulence, we have characterized the DSF deficient mutant of X. oryzae pv. oryzicola. Mutant analysis by expression analysis, radiolabelled iron uptake studies and growth under low-iron conditions indicated that DSF positively regulates ferric iron uptake. Further, the DSF deficient mutant of X. oryzae pv. oryzicola exhibited a reduced capacity to use ferric form of iron for growth under low-iron conditions. Exogenous iron supplementation in the rice leaves rescued the in planta growth deficiency of the DSF deficient mutant. These data suggest that DSF promotes in planta growth of X. oryzae pv. oryzicola by positively regulating functions involved in ferric iron uptake which is important for its virulence. Our results also indicate that requirement of iron uptake strategies to utilize either Fe(3+) or Fe(2+) form of iron for colonization may vary substantially among closely related members of the Xanthomonas group of plant pathogens.

Transcriptome analysis reveals a common adaptive transcriptional response of Candida glabrata to diverse environmental stresses.

Candida glabrata, an opportunistic fungal pathogen, causes superficial and life-threatening infections in humans. In the host microenvironment, C. glabrata encounters a variety of stresses, and its ability to cope with these stresses is crucial for its pathogenesis. To gain insights into how C. glabrata adapts to adverse environmental conditions, we examined its transcriptional landscape under heat, osmotic, cell wall, oxidative, and genotoxic stresses using RNA sequencing and reveal that C. glabrata displays a diverse transcriptional response involving ∼75% of its genome for adaptation to different environmental stresses. C. glabrata mounts a central common adaptation response wherein ∼25% of all genes (n = 1370) are regulated in a similar fashion at different environmental stresses. Elevated cellular translation and diminished mitochondrial activity-associated transcriptional signature characterize the common adaptation response. Transcriptional regulatory association networks of common adaptation response genes revealed a set of 29 transcription factors acting as potential activators and repressors of associated adaptive response genes. Overall, the current work delineates the adaptive responses of C. glabrata to diverse environmental stresses and reports the existence of a common adaptive transcriptional response upon prolonged exposure to environmental stresses.

Atypical regulation of virulence-associated functions by a diffusible signal factor in Xanthomonas oryzae pv. oryzae.

In Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, a secreted fatty acid signaling molecule known as diffusible signal factor (DSF) is required for virulence and growth on low-iron medium. To identify other virulence-associated traits that are regulated by DSF in this pathogen, we have performed microarray analysis of transcriptional changes between the wild type and DSF-deficient mutants of X. oryzae pv. oryzae. Expression of genes that encode secreted hydrolytic enzymes, motility, and chemotaxis functions are negatively regulated by DSF while functions involved in adhesion and biofilm formation are positively regulated. Enzymatic assays for hydrolytic enzymes as well as assays for chemotaxis, motility, attachment, and biofilm formation corroborate these findings. These results demonstrate that, in X. oryzae pv. oryzae, DSF-mediated cell-to-cell signaling coordinates transition from solitary to biofilm lifestyle by promoting expression of attachment functions and negatively regulating expression of motility functions. This is in contrast to X. campestris pv. campestris, a pathogen of crucifers, wherein the DSF system positively regulates motility functions and negatively regulates biofilm formation. These results indicate that virulence-associated functions can be regulated in a completely contrasting fashion by the same signaling system in very closely related bacteria.

Transcriptomic analysis reveals global and temporal transcription changes during Candida glabrata adaptation to an oxidative environment.

The ability to survive host-elicited oxidative stress is critical for microbial pathogens to cause infection. The human fungal pathogen C.glabrata can tolerate high levels of oxidative stress and proliferate inside phagocytes. Previous studies had successfully identified a transcription response to oxidative stress including induction of a core set of detoxification genes. However, the findings only represent an early snapshot of a highly dynamic process lacking temporal resolution. Here, we compare the transcriptome of C. glabrata at various points after exposure to hydrogen peroxide in order to study its adaptation to an oxidative environment. Our results reveal global and temporal gene expression changes during an immediate response; up-regulating genes related to peroxide detoxification, while down-regulating genes essential for growth. As cells adapt to the oxidative environment, a dramatic transcriptome reprogramming occurred to restore key cellular functions, protein homeostasis and biosynthesis of trehalose, carbohydrate, fatty acid and ergosterol. Interestingly, biofilm and drug transporter genes as well as many genes implicated in virulence, were induced during the adaptation stage. Our finding, therefore, suggests a role of oxidative stress adaptation in promoting virulence and drug resistance traits of C. glabrata during infection.

Xanthoferrin, the α-hydroxycarboxylate-type siderophore of Xanthomonas campestris pv. campestris, is required for optimum virulence and growth inside cabbage.

Xanthomonas campestris pv. campestris causes black rot, a serious disease of crucifers. Xanthomonads encode a siderophore biosynthesis and uptake gene cluster xss (Xanthomonas siderophore synthesis) involved in the production of a vibrioferrin-type siderophore. However, little is known about the role of the siderophore in the iron uptake and virulence of X. campestris pv. campestris. In this study, we show that X. campestris pv. campestris produces an α-hydroxycarboxylate-type siderophore (named xanthoferrin), which is required for growth under low-iron conditions and for optimum virulence. A mutation in the siderophore synthesis xssA gene causes deficiency in siderophore production and growth under low-iron conditions. In contrast, the siderophore utilization ΔxsuA mutant is able to produce siderophore, but exhibits a defect in the utilization of the siderophore-iron complex. Our radiolabelled iron uptake studies confirm that the ΔxssA and ΔxsuA mutants exhibit defects in ferric iron (Fe3+ ) uptake. The ΔxssA mutant is able to utilize and transport the exogenous xanthoferrin-Fe3+ complex; in contrast, the siderophore utilization or uptake mutant ΔxsuA exhibits defects in siderophore uptake. Expression analysis of the xss operon using a chromosomal gusA fusion indicates that the xss operon is expressed during in planta growth and under low-iron conditions. Furthermore, exogenous iron supplementation in cabbage leaves rescues the in planta growth deficiency of ΔxssA and ΔxsuA mutants. Our study reveals that the siderophore xanthoferrin is an important virulence factor of X. campestris pv. campestris which promotes in planta growth by the sequestration of Fe3+ .