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CSF levels of Elongation Factor Tu is associated with increased mortality in human Streptococcus pneumoniae meningitis

ABSTRACT: Background Mortality from bacterial meningitis, predominately caused by Streptococcus pneumoniae, exceeds 50% in low and middle income countries in sub-Saharan Africa with high HIV prevalence. Underlying causes of high mortality are poorly understood. We examined the host and pathogen proteome in adults with proven pneumococcal meningitis (PM), testing if differentially expressed proteins in CSF were implicated in outcome. Materials/methods CSF proteomes were analysed by quantitative Mass-Spectrometry. Spectra were identified using the Swissprot human and TIGR4 pneumococcal protein libraries. Proteins were quantitated and analysed against clinical outcome. Unique proteins in PM were identified against published normal CSF proteome. Random-Forest models were used to test for protein signatures discriminating outcome. Proteins of interest were tested for their effects on growth and opsonophagocytic killing of S. pneumoniae. Results CSF proteomes were available for 57 Adults with PM (median age 32 years, 60% male, 70% HIV-1 co-infected, mortality 63%). 360 individual human and 23 pneumococcal proteins were identified. Of the human protein hits, 30% were not expressed in normal CSF, and these were strongly associated with inflammation and primarily related to neutrophil activity. No human protein signature predicted outcome. However, expression of the essential S. pneumoniae protein Elongation Factor Tu (EF-Tu) was significantly increased in CSF of non-survivors (False Discovery Rate (q) <0.001). Expression of EF-Tu was negatively co-correlated against expression of Neutrophil defensin (r 0.4 p p<0.002), but not against complement proteins C3 or Factor H. In vitro, addition of EF-Tu protein impaired S. pneumoniae neutrophil killing when osponised with CSF. Conclusions Excessive CSF levels of S. pneumoniae EF-Tu protein was associated with reduced survival in human meningitis. Preliminary studies show EF-Tu inhibits neutrophil mediated killing of S. pneumoniae in CSF. Further functional studies are required to better understand the mechanistic role of EF-Tu during PM.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cerebrospinal Fluid

SUBMITTER: Philip Brownridge

LAB HEAD: Emma Wall

PROVIDER: PXD021268 | Pride | 2020-11-09

REPOSITORIES: pride

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			Action	DRS
	EmmaWall_batch1_280515_06_sample1.raw	Raw
	EmmaWall_batch1_280515_06_sample1_mzcorrected.mzML	Mzml
	EmmaWall_batch1_280515_06_sample1_mzcorrected.mzid.gz	Mzid
	EmmaWall_batch1_280515_07_sample2.raw	Raw
	EmmaWall_batch1_280515_07_sample2_mzcorrected.mzML	Mzml

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Project description:We applied genomic array footprinting (GAF) in the search for genes of S. pneumoniae essential during the establishment and progression of experimental meningitis. Four libraries with a total of 6,000 independent TIGR4 marinerT7 transposon mutants were injected intra-cisternally in rabbits, and cerebrospinal fluid (CSF) was collected after three, nine, and fifteen hours. Microarray analysis of mutant-specific probes from CSF samples and inocula identified 82 genes of which mutants had become attenuated and eleven genes of which mutants had become enriched during infection. Screening results particularly point to an essential role for capsular polysaccharides (cps), nutrient uptake, and metabolism in meningitis. Detailed study on directed mutants of a subset of sixteen GAF targets in an experimental model of rat meningitis revealed that ten were significantly attenuated or enriched during competitive infection. Furthermore, we showed that mutants of adenylosuccinate synthetase (purA), flavodoxin (fld), and the substrate binding protein (livJ) of a branched chain amino acid ABC-transporter were essential for full blown meningitis in a mono-infection setup of rat meningitis. Overall, the GAF screen revealed the general restraint on nutrients encountered by the pneumococcus in CSF, while, except for cps genes, no ‘classic’ virulence factors appeared to be required for infection. This knowledge will contribute to a better understanding of the molecular pathogenesis of pneumococcal meningitis. The rabbit meningitis model was performed as previously described by Østergaard (PMID: 9661008 ). Briefly, 10E6 CFU of a S. pneumoniae mutant library was suspended in 20 µl beef-broth and injected into cisterna magna of four groups of four outbred New Zealand White rabbits of app. 2.5 kg (in one group one rabbit died). CSF (0.3 ml) samples were repeatedly obtained from the same rabbit by aspiration from indwelling spinal- and arterial cannules. Samples were collected before pleocytosis developed (three hrs), at the time of maximal pleocytosis (nine hrs), and at a time of slowing bacterial growth and abating pleocytosis (fifteen hrs ). Until further processing in the GAF protocol, CSF and inocula were stored with 15% glycerol at -80ºC. The GAF technology was performed as described previously (PMID: 17261526 ). Briefly, stored inocula and CSF obtained during the experimental rabbit meningitis experiments were defrosted, diluted in GM17 medium supplemented with spectinomycin, and grown to mid-log phase. Chromosomal DNA was isolated, digested with AluI endonuclease, and used as a template for in vitro transcription initiated from the T7 promoters of the marinerT7 transposon that is present in each mutant. After removal of template DNA by DNAseI treatment, mutant-specific T7 RNA was reverse transcribed in the presence of fluorescent Cy3/Cy5-labeled dUTP nucleotides. Labeled Cy3/Cy5-cDNA from the CSF samples and oppositely labeled Cy5/Cy3 cDNA from the inocula were combined, purified, and washed by ultrafiltration. The cDNA was hybridized to pneumococcal microarrays containing, 2,087 ORFs of Streptococcus pneumoniae TIGR4 and 70-mer oligo's specific for the non-homologous ORFs in the R6, D39, 23F, INV104B, INV200, OXC141, and G54 strains, all spotted in duplicate (PMID: 18174343) . Dual channel array images were acquired on a GenePix 4200AL microarray scanner (Axon Instruments, Union City, CA). Microarray image files were analyzed with GenePix Pro software (Axon Instruments, Union City, CA). Spots were screened visually to identify those of low quality and removed from the data set prior to analysis. A net mean intensity filter based on hybridization signals obtained with R6-specific spots was applied in all experiments. Slide data were processed and normalized using MicroPreP (PMID: 15907200). Further analysis was performed using a CyberT implementation of the Student’s t test (cybert.microarray.ics.uci.edu). This web-based program lists the ratios of all intra-replicates (duplicate spots) and inter-replicates (different slides), the mean ratios per gene, and standard deviations and (Bayesian) p values assigned to the mean ratios. For identification of conditionally essential genes, only genes with a minimum of 6/8 (for groups of four rabbits) or 5/6 for the group of three rabbits) reliable measurements and a Bayesian p-value < 0.001 were included. Further selection criteria were applied, as only mutants displaying an average fold-change of >2.5 at a minimum of two time-points, or >4.0 at one time-point were accepted as significantly attenuated or enriched. The microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) database (www.ncbi.nlm.nih.gov/geo/) under GEO Series accession number ...

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CSF levels of Elongation Factor Tu is associated with increased mortality in human Streptococcus pneumoniae meningitis

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