Dataset Information

The architecture and pathogenesis of Pseudomonas-Candida biofilms in infections and their influence on the microenvironment

ABSTRACT: Interkingdom Pseudomonas-Candida biofilms is a human pathogen associated with acute and chronic infection. This study examine hoe their structural organization influences their in vivo microenvironment, which in turns affects the interaction of Pseudomonas-Candida biofilms with host immune responses. NCI-H292 grown P. aeruginosa biofilms with and without C. albicans biofilms were used for proteomic analysis. Biofilms were demonstrated in all groups which have 357 proteins from proteomic analysis including i) proteins in PA+CA were higher than PA (76 proteins, such as sigma factor AlgU negative regulatory protein; MucA) and ii) proteins in PA+CA were lower than PA (281 proteins, including alginate genes such as Alginate biosynthesis transcriptional regulatory protein; AlgB). Although most of Pseudomonas proteins in PA+CA were lower than PA due to the lower initial bacteria in preparation of mixed-organism biofilms, mucA overexpressed in PA+CA indicating that high alginate production that transcriptionally controlled by algU-mucABCD genes. These results demonstrated that Pseudomonas-Candida biofilms produced alginate more compared to Pseudomonas biofilms on lungs.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Candida Albicans (yeast)

SUBMITTER: Jiradej Makjaroen

LAB HEAD: Asada Leelahavanichkul

PROVIDER: PXD034528 | Pride | 2022-10-13

REPOSITORIES: Pride

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Publications

Rapid Synergistic Biofilm Production of Pseudomonas and Candida on the Pulmonary Cell Surface and in Mice, a Possible Cause of Chronic Mixed Organismal Lung Lesions.

Phuengmaung Pornpimol P Mekjaroen Jiradej J Saisorn Wilasinee W Chatsuwan Tanittha T Somparn Poorichaya P Leelahavanichkul Asada A

International journal of molecular sciences 20220816 16

Due to the possible co-presence of Pseudomonas aeruginosa and Candida albicans (the most common nosocomial pathogens) in lungs, rapid interkingdom biofilm production is possible. As such, PA+CA produced more dominant biofilms on the pulmonary epithelial surface (NCI-H292) (confocal fluorescent extracellular matrix staining) with dominant psl upregulation, as demonstrated by polymerase chain reaction (PCR), after 8 h of experiments than PA alone. With a proteomic analysis, rh ...[more]

PMID: 36012475

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Project description:Candida spp. are commensal opportunistic fungal pathogens that often colonize and infect mucosal surfaces of the human body. Candida, along with other microbes in the microbiota, generally grow as biofilms in a polymicrobial environment. Due to the nature of cellular growth in a biofilm (such as production of a protective extracellular matrix) and the recalcitrance of biofilms, infections involving biofilms are very difficult to treat with antibiotics and perpetuate the cycle of infection. The two most commonly isolated Candida spp. from Candida infections are Candida albicans and Candida glabrata, and the presence of both of these species results in increased patient inflammation and overall biofilm formation. This work aims to investigate the interspecies interactions between C. albicans (Ca) and C. glabrata (Cg) in co-culture through transcriptome analysis over the course of biofilm growth. We report that during co-culture, lipid biosynthesis and transporter genes were significantly modulated in both Ca and Cg. Differentially expressed genes in Ca during co-culture growth included putative transporter genes (C2_02180W_A and C1_09210C_B; up-regulated), amino acid biosynthesis (ARO7; up-regulated most in Ca:Cg 1:3), and lipid-related genes (LIP3 and IPT1; down-regulated). Differentially expressed genes in Cg in co-culture included putative transmembrane transporters (CAGL0H03399g and CAGL0K04609g; up-regulated), an oxidative stress response gene (CAGL0E04114g; down-regulated most in Ca:Cg 1:3), genes involved in the TCA cycle (LYS12 and CAGL0J06402g; down-regulated), and several genes involved in cell wall/membrane biosynthesis (SEC53, GAS2, VIG9; down-regulated). Additionally, confocal microscopy was utilized for membrane lipid analysis between monoculture and co-culture biofilms. Through filipin-stained lipid analysis, we found that there was a significant increase in cell membrane lipid content in Ca:Cg 1:3 biofilms compared to Ca and Ca:Cg 3:1 biofilms. These results suggest substantial modifications of both cell wall, cell membrane, and transporters in both Ca and Cg during the time course of co-culture growth, which allows for increased biofilm formation and virulence in Candida co-culture biofilms.

Project description:Bacteria growing in biofilms are physiologically heterogeneous, due in part to their adaptation to local environmental conditions. Here, we characterized the local transcriptional responses of Pseudomonas aeruginosa growing in biofilms by using microarray analysis of isolated biofilm subpopulations. The results demonstrated that cells at the top of the biofilms had high mRNA abundances for genes involved in general metabolic functions, while mRNAs for these housekeeping genes were low in cells at the bottom of the biofilms. Selective GFP labeling showed that cells at the top of the biofilm were actively dividing. However, the dividing cells had high mRNAs levels for genes regulated by the hypoxia induced regulator, Anr. Slow-growing cells deep in the biofilms had little expression of Anr-regulated genes and may have experienced long-termanoxia. Transcripts for ribosomal proteins were primarily associated with the metabolically active cell fraction, while ribosomal RNAs were abundant throughout the biofilms, indicating that ribosomes are stably maintained even in slowly growing cells. Consistent with these results was the identification of mRNAs for ribosome hibernation factors (rmf and PA4463) at the bottom of the biofilms. A P. aeruginosa M-bM-^HM-^Frmf strain had increased uptake of the membrane integrity stain, propidium iodide. Using selective GFP labeling and cell sorting, we showed that the dividing cells were more susceptible to tobramycin and ciprofloxacin than the dormant subpopulation. The results demonstrate that in thick P. aeruginosa biofilms, cells are physiologically distinct spatially, with cells deep in the biofilm in a viable but antibiotic-tolerant slow-growth state. 52-hour Pseudomonas aeruginosa TSA colony biofilms were cryoembedded, thin sectioned, and laser dissected (LCM) to obtain samples from the top and bottom 50 M-BM-5m of the biofilms. 9 sections per biofilm were pooled. RNA was extracted with the RNeasy Micro kit, Turbo DNase treated, poly(A) tailed, and amplified using the Quantitect WTA kit. After clean up, the resulting product was fragmented and end labeled before hybridization.

Project description:Pseudomonas aeruginosa is one of the most frequent pathogen dominant in complicated urinary tract infections (UTI). To unravel the adaptation strategies of P. aeruginosa to the conditions in the urinary tract and to define the underlying regulatory network an artificial growth system mimicking the conditions in the urinary tract was established. Transcriptome analyses were used to investigate the physiological status of P. aeruginosa under this conditions. We performed comparisons to identify genes induced under artificial urinary tract conditions to unravel the adaptive strategies and the underlying regulatory network used by Pseudomonas aeruginosa during urinary tract infections using Affimetrix GeneChips. Pseudomonas aeruginosa wild type strain PAO1 was grown in an artificial in vitro growth system mimicking the conditions in the urinary tract. Therefore, biofilms were grown on the surface of membrane filters placed on agar plates at 37 Â°C up to the late logarithmic state under aerobic and anaerobic conditions (incubated in an anaerobic beanch). An artificial urine medium (AUM) simulating the averaged urine of an human adult was used as nutrient souce. 10-fold diluted Luria Bertani (LB)-medium was used as reference medium. For growth under oxygen depletion the media were supplemented with 50 mM KNO3 to sustain anaerobic respiration. The biofilms were harveted at this time points and resuspsended in 0.9% (w/v) NaCl. The OD578 of biofilm suspension was 0.8 for all tested conditions. First comparison: Identification of genes induced or repressed under aerobic conditions in the P. aeruginosa wild type PAO1. Here we compared the transcriptome profile of P. aeruginosa PAO1 grown aerobically for 18 h to the late logarithmic phase in biofilms on AUM with the transcriptome profile of the PAO1 strain, which was grown aerobically for 18 h to the late logarithmic phase in biofilms on 10-fold diluted LB. Second comparison: Identification of genes induced or repressed under anaerobic conditions in the P. aeruginosa wild type PAO1. Here we compared the transcriptome profile of P. aeruginosa PAO1 grown anaerobically for 2 days up to the late logarithmic phase in biofilms on AUM supplemented with 50 mM nitrate with the transcriptome profile of the PAO1 strain, which was grown anaerobically for 2 days up to the late logarithmic phase in biofilms on 10-fold diluted LB supplemented with 50 mM nitrate.

			Action	DRS
	H292_ATCC_26042022.pdStudy	Other
	H292_ATCC_26042022.pdStudy.bak	Other
	H292_Set1_F.msf	Msf
	H292_Set1_F.msfView	Msf
	H292_Set1_F.pdResult	Other

Dataset Information

The architecture and pathogenesis of Pseudomonas-Candida biofilms in infections and their influence on the microenvironment

Dataset's files

Publications

Rapid Synergistic Biofilm Production of <i>Pseudomonas</i> and <i>Candida</i> on the Pulmonary Cell Surface and in Mice, a Possible Cause of Chronic Mixed Organismal Lung Lesions.

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