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Corynebacterium diphtheriae proteome adaptation to cell culture medium and serum

ABSTRACT: ost-pathogen interactions are often studied in vitro using primary or immortal cell lines. This set-up avoids ethical problems of animal testing and has the additional advantage of lower costs. However, the influence of cell culture media on bacterial growth and metabolism is not considered or investigated in most cases. To address this question growth and proteome adaptation of Corynebacterium diphtheriae strain ISS3319 were investigated in this study. Bacteria were cultured in standard growth medium, cell culture medium and fetal calf serum. Mass spectrometric analyses and label-free protein quantification hint to an increased bacterial pathogenicity when grown in cell culture medium as well as an influence of the growth medium on the cell envelope.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Corynebacterium Diphtheriae Bv. Mitis Str. Iss 3319

DISEASE(S): Diphtheria

SUBMITTER: Jens Möller

LAB HEAD: Andreas Burkovski

PROVIDER: PXD024381 | Pride | 2021-03-12

REPOSITORIES: Pride

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Project description:Neisseria meningitidis remains an important cause of septicemia and meningitis. One of its major virulence traits is its ability to avoid killing by human serum. In the present work, the effect of growth phase (exponential versus stationary) and growth medium (THB versus Catlin) on normal human serum (NHS) sensitivity was assessed on two N. meningitidis serogroup B strains (MC58 and M982). Both strains were found to be dramatically more resistant to 20% NHS killing at early exponential phase than at stationary phase (73-100% survival after 1 to 2 hours of growth compared to less than 10% survival after 7 hours of growth). This growth phase effect was detected only when a rich medium (THB) was used while no such effect was observed using Catlin medium. KDO (2-Keto-3-deoxyoctulosonic acid) and sialic acid content were measured on serum-resistant and serum-sensitive bacteria and were found comparable, indicating that differences in LPS and capsule expression were not at the origin of the observed phenotypes. Transcriptome profiling using a PCR-array was used to compare serum-resistant and serum-sensitive bacteria. A set of 255 genes was found to be differentially expressed with 159 genes up-regulated in serum-resistant bacteria, among them 12 genes involved in glucose catabolism and 22 are known virulence factors. Overall, this study shows that serum-resistant phenotype of N. meningitidis could be obtained through modulating growth conditions. The nature of growth media and growth phases have a major impact on the ability of N. meningitidis to resist to NHS killing. Consequently, the present work underlines the critical importance of carefully controlling those parameters in any studies aimed at investigating the mechanisms and factors involved in N. meningitidis serum-resistance. All experiments were performed in dye swap. These experiments were performed with 2 strains of N. meningitidis serogroup B. In one set of experiments, the gene expression profile of bacteria cultured in THB medium during 7h was compared with the corresponding profile of bacteria cultured during 1h in the same medium. In a second set of experiments, the gene expression profile of 1 strain cultured in THB medium during 7h was compared with the corresponding profile of the same strain cultured in Catlin medium during 7h. 3 biological replicates were performed for each comparison.

Project description:Analysis of human primary macrophages after live Lactobacillus rhamnosus GG (LGG) or LC705 stimulation for 6h and 24h. The results reveal novel mechanisms for probiotics-induced activation of the healthy human innate immune system. Macrophages are phagocytic cells of the innate immune system that perform sentinel functions to initiate appropriate responses to surrounding stimuli. Macrophages that reside at mucosa encounter ingested bacteria. Our understanding of the responses elicited by nonpathogenic bacteria in human innate immune system is limited. Lactobacillus are nonpathogenic bacteria commonly used in food and as supplements with health-promoting probiotic potential. In this study, we have utilized global gene expression profiling to compare the responses of human primary macrophages to two closely related, well-characterized L. rhamnosus strains LGG and LC705. Our results demonstrate that live LGG and LC705 induced quantitatively different gene expression in macrophages. A gene ontology analysis revealed functional similarities and differences in responses to LGG and LC705. Both LGG and LC705 induced IL-1b production in macrophages that required caspase-1 activity. LC705 but not LGG induced a strong type I IFN-dependent gene activation that correlated with the ability of LC705 to prevent influenza A virus replication and production of viral proteins in macrophages. Differentiated 7d human primary macrophages from 18 healthy individuals were stimulated with either live L. rhamnosus GG (LGG) or LC705 for 6 h and 24 h in RPMI medium containing penicillin and streptomycin. As a control, macrophages were stimulated with the medium only. The experiment was performed three times, each time with cells from six different individuals (samples 1-3). For each experiment, macrophages were stimulated separately as described, and macrophages from different donors were pooled after each stimulation experiment. Extracted RNA was hybridized to AffymetrixM-BM-. U133-plus2.0 GeneChipM-BM-. arrays.

Project description:Approximately 2x106 CL1-5 and H23 cells were seeded on 10-cm dishes with RPMI-1640 medium containing 10% fetal bovine serum. After the cells covered 80% of the dish, cells were serum-starved for 24 h in RPMI-1640 medium. CL1-5 and H23 cell lysates were harvested, trypsinized and analyzed by using LTQ-Orbitrap XL and LTQ-Orbitrap Discovery, respectively. Halobacterium salinarum NRC-1 (ATCC700922) starter culture was prepared by inoculating a single colony in 5 mL of Halobacterium medium containing trace metals (CM+) and grown at 37 °C with shaking at 225 rpm for one week. An aliquot of 300 μL of the culture was spread on 15-cm diameter CM+ plates containing 2% of agarose. After 7~10 days of incubation at 37 °C, the GVs from cells on 10 plates were harvested using the centrifugation facilitated flotation method as previously described except the GVs were washed for a total of 5 times instead of the suggested 3 times to remove nonspecific bound proteins.(29) After collecting the gas vesicles in the primary cell lysate, the cell membrane and residual GV were removed by ultracentrifugation at 53 000× g, 4 °C for 16 h twice to obtain the GV-depleted lysate (GVD). GVD lysate was harvested, trypsinized and analyzed by using LC-MS/MS ( LTQ-Orbitrap XL). The H. salinarum strain NRC-1 was grown in complex medium (CM+) at 37 oC with shaking (200 rpm) to an OD600 of 0.5 (early exponential phase. The cultures were then subjected to an additional hour of incubation at 37 oC for preparation of proteins. The total lysate was harvested, trypsinized and analyzed by using LC-MS/MS (LTQ-Orbitrap Discovery). All MS raw data in Thermo XCalibur (version 2.0.7) binary format were converted to the mzXML open data format using a modified version of the ReAdW program. SEQUEST (version 27) was applied to search the MS/MS spectra against the forward and reverse (decoy) protein sequences of H. salinarum (2,427 proteins; EMBL-EBI Integr8, www.ebi.ac.uk/integr8/EBI-Integr8-HomePage.do) or human (38,425 proteins) plus bovine (22,863 proteins) (GenBank reference sequences, www.ncbi.nlm.nih.gov). SEQUEST results were further processed using the Trans Proteomic Pipeline.

Project description:PrfA activity was studied in L. monocytogenes strain EGD and in an isogenic prfA deletion mutant (EGDΔprfA) carrying multiple copies of the wild-type prfA or the mutant prfA* gene (strains EGDΔprfApPrfA and EGDΔprfApPrfA*) after growth in brain heart infusion (BHI), Luria-Bertani broth (LB) or a defined minimal medium (MM) supplemented either with one of the three PTS-carbohydrates, glucose, mannose and cellobiose, or the non-PTS carbon source glycerol. Low PrfA activity was observed in the wild-type EGD strain in BHI and LB with either of these carbon sources, while PrfA activity was high in minimal medium in presence of glycerol but significantly reduced in presence of cellobiose. The strains expressing the prfA and prfA* gene under the prfA promoters, P1 and P2, produced equally large amounts of PrfA protein and high PrfA activity was observed in strain EGDΔprfApPrfA* under all growth conditions. In contrast, high PrfA activity in strain EGDΔprfApPrfA was only observed when this strain was cultured in BHI but not in LB or MM (in presence of either carbon source). A ptsH mutant (lacking a functional HPr) was able to grow in BHI suggesting that growth of L. monocytogenes in this culture medium is supported by carbon sources whose uptake and metabolism are independent of the PTS pathway. However, this mutant was unable to grow in LB and MM regardless which of the four carbon sources was added, suggesting that uptake of the used carbohydrates and the catabolism of glycerol depend fully on the functional common PTS pathway. Furthermore, the growth rates of L. monocytogenes are strongly reduced in presence of large amounts of PrfA protein when growing MM but less in LB and only slightly in BHI. The expression profiles of the genes encoding PTS permeases were determined in the three strains under various growth conditions. The data suggest that PrfA activity correlates with the expression level and the phosphorylation state of specific PTS permeases. This SuperSeries is composed of the following subset Series: GSE12143: Listeria monocytogenes EGD after growth BHI vs. LB vs. MM GSE12145: Listeria monocytogenes EGDΔprfApPrfA and EGDΔprfApPrfA* compared to the wild type strain EGD GSE12146: Listeria monocytogenes EGD and EGD-e

			Action	DRS
	BHI_R1.msf	Msf
	BHI_R1.raw	Raw
	BHI_R2.msf	Msf
	BHI_R2.raw	Raw
	BHI_R3.msf	Msf

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Corynebacterium diphtheriae proteome adaptation to cell culture medium and serum

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