Supplementary online material
Pro-inflammatory cytokines can act as intracellular modulators of commensal bacterial virulence
Jafar Mahdavi1‡, Pierre-Joseph Royer1, Hong S Sjölinder7, Sheyda Azimi1, Tim Self2, Jeroen Stoof1, Lee M. Wheldon1, Kristoffer Brännström6, Raymond Wilson3, Joanna Moreton3, James W. B. Moir4, Carina Sihlbom5, Thomas Borén6, Ann-Beth Jonsson7, Panos Soultanas8‡, Dlawer A.A. Ala’Aldeen1‡
1School of Molecular Medical Sciences, The University of Nottingham, Nottingham NG7 2RD, UK, 2Institute of Cell Signaling, The University of Nottingham, Nottingham NG7 2UH, UK, 3Deep Seq, Centre for Genetics and Genomics, The University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, UK, 4Department of Biology, University of York, Heslington, York, YO10 5YW, UK. 5Proteomics Core Facility, Sahlgrenska Academy, University of Gothenburg, Box 413, SE-405 30 Gothenburg, Sweden, 6Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden. 7Department of Genetic, Microbiology and Toxicology (GMT), Stockholm University, 109 61 Stockholm, Sweden, 8School of Chemistry, The University of Nottingham, Nottingham NG7 2RD, UK.
‡ To whom correspondence should be addressed.
Jafar Mahdavi
Phone: +44-(0)115 8468925
Fax: +44-(0)115 823 0759
E-mail:
Panos Soultanas
Phone: +44 (0)115 9513525
Fax: +44 (0)115 8468002
E-mail:
Dlawer Ala’Aldeen
Phone: +44-(0)115 8230748
Fax: +44-(0)115 823 0759
E-mail:
Specific Discussion
Over the course of infection by a pathogen, the immune system rapidly activates a number of defence mechanisms, characterized by the increased production of immune mediators (i.e., cytokines). This reaction, known as the innate immune response, is mediated by pattern-recognition receptors that detect conserved structures found in a broad range of pathogens [1]. N. meningitidis is usually a commensal bacterium of the nasopharynx. Factors that lead to the invasion of the bloodstream, often followed by the crossing of the blood-brain barrier and meningitis, are likely partly host- and partly bacterium-dependent. Hence, well-equilibrated gene regulation systems must exist, allowing the bacteria to monitor the environment and survive sufficiently long, without killing their host, to ensure an effective transmission of the species. During the commensal state, most of the dividing bacteria belong to the same antigenic type and express lower levels of virulence genes. Most likely, a peak of pathogenic status (i.e., bacteraemia followed by meningitis) is reached when the bacteria sense danger caused by a hyper-inflamed environment (illustrated in Fig. 7b, main paper).
Tumor necrosis factor alpha (TNF-α) and IL-8 are pro-inflammatory cytokines that have numerous biological activities [2] and are believed to play important roles not only in host defence [2,3] but also in some of the pathological squeal associated with various bacterial infections [4,5]. Consequently, bacteria developed sophisticated molecular machines for accurate sensing of the host environment and efficient uptake of host proteins which then modulate the expression of bacterial genes required for virulence and survival within the host.
Cytokines contain lectin-like carbohydrate domains which are spatially distinct from cytokine-receptor binding sites [6]. Our investigation of the binding properties of a series of glycosyltransferase-deficient mutants (∆pglC and a ∆pglC/L double mutant) suggest that the Nm-cytokine binding is mediated partly by glycan moieties and by protein-protein interactions (Fig. 2 main paper, S3-Fig.). This observation is in agreement with the findings of Estabrook et al., who showed that mannose-binding lectin also binds to the non-glycosylated outer membrane proteins Opa and PorB of Nm in a carbohydrate-independent manner [7]. The data presented here show that binding of TNF-α or IL-8 to Nm is mediated by pilus assembly (i.e., PilQ and PilE proteins) and that the virulence properties of Nm are enhanced as a consequence of TNF-α or IL-8 binding and uptake (Fig. 3 main paper). The ingested cytokines directly bind to genomic DNA (Fig. 5 main paper) and consequently regulate the expression of several genes (S7 and S7 Deep sequencing Figures).
These findings have numerous implications in terms of our understanding of Tfp biogenesis/function and provide a useful groundwork for the precise functional characterization of the PilE, PilQ and other pili proteins.
N. meningitidis has evolved to become commensal within the nasopharyngeal epithelium. Once the bacterium comes into contact with the host epithelium, the program of gene modulation would remain more-or-less unaffected until substantial environmental variations occur. This implies a transient interaction with human epithelial cells and a tendency for the bacteria to reorganize their effectors and, consequently, transcription/translation profiles to rapidly adapt to new environmental conditions and, depending on the extent of environmental alterations, change towards a pathogenic status (Illustrated in Fig. 7b main paper).
In conclusion, our findings provide a mechanism to explain the frequent development of meningitis in patients with an intense and protracted inflammatory response. Further research comparing the nature of hypervirulent lineages may elucidate the extent to which this feature contributes to the epidemiological distinctiveness of meningococcal infections.
Supplementary material 1a: The effect of human cytokines on N. meningitidis growth.
Several studies have shown that pro-inflammatory cytokines enhance the growth of pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus and Acinetobacter spp. Meduri et al. provided additional evidence for a newly described pathogenic mechanism for bacterial proliferation in the presence of exaggerated and protracted inflammation [8]. Here, we examined the effect of cytokines on the growth of Nm strain MC58. The results showed no changes in bacterial growth after incubation with either IL-8 or TNF-α (S1-Fig. a,b).
S1a-Figures. Bacterial growth curves for N. meningitidis. Bacterial growth in DMEM was determined by measuring the OD at 600 nm following incubation at 37oC, 5% CO2. Wild-type MC58 was either left untreated (CT) or treated with TNF-α (a) or IL-8 (b) at 20 or 40 ng/ml. All bacterial samples grew from an equal starting OD600 of 0.01. The time shown refers to the duration of the incubation and the marked 9 h time point (circle) refers to the harvesting time point for RNA purification.
Supplementary material 1b: Binding of meningococcal clinical isolates to TNF-α and IL-8.
To investigate the binding ability of other sero-groups of N. meningitidis strains (serogroups B and Y) to TNF-α and IL-8, 10 N. meningitidis clinical isolates were analysed in comparison with MC58 strain (serogroup B). The results clearly indicate that all meningococcal strains bind to both TNF-α and IL-8, albeit to variable degrees, irrespective of their invasiveness, phenotypic characteristics or geographical distribution (Fig. a, b).
S1b-Figure: N. meningitidis clinical isolates bind to TNF-α and IL-8. N. meningitidis strain MC58, and 5 clinical isolates from each serogroup (B and Y) were digoxigenin labeled and examined the interaction with TNF-α and IL-8 coated ELISA plate. All the strains were capable of binding to both TNF-α and IL-8, although at different degree. The data represent the mean (OD) at wavelength of 405 nm ± SEM (error bars) of a sample tested in triplicate. Experiments were repeated three times, with consistent results.
Supplementary material 2. Glycosylation of PilE. The O-linked glycans are associated covalently with the hydroxyl groups of serines or threonines. Type IV pilin (PilE) of pathogenic Neisseria was one of the first examples of an O-glycosylated glycoprotein [9]. Two types of O-linked trisaccharide have been identified in Nm pili (the specific type expressed depending on the host strain): Gal-β1, 4-Gal-α1, 3-DATDH (DATDH represents 2, 4-diacetamido-2, 4, 6-trideoxyhexose), [10] or Gal-β1, 4-Gal-α1, 3-GATDH (GATDH represents 2-glyceramido-4-acetamido-2, 4, 6-trideoxyhexose) [11] (S2-Table ). An additional truncated O-linked disaccharide (Gal-α1, 3GlcNAc), is also present in Nm strain 8013 [12].
Protein target / O-Tase transferase / Amino acids modified / Glycan transferred / Ref.Pilin (PilE) / PglL / Promiscuous; multiple O-linked repeats / Serine 63 / / [10]
/ [11]
/ [12]
S2-Table: Structure of meningococcal O-linked glycosylated pilin. Adopted from [13] *Key to symbols:
A novel O-linked trisaccharide substituent, which has not previously been identified as a constituent of glycoproteins, is present within a peptide spanning amino acid residues 45 to 73 of the PilE molecule. This structure contains a terminal 1-4-linked digalactose moiety that is covalently linked to a 2,4-diacetamido-2,4,6-trideoxyhexose sugar, which is directly attached to pilin [10].
Many cytokines possess lectin-like activity that may be essential for the expression of their full biological activities. Here, we focused on the relevance of the lectin-like activity of cytokines in mediating Nm binding, using TNF-α and IL-8 as illustrative examples.
It is well established that IL-8 and TNF-α interact with heparan sulfate proteoglycans [14]. IL-8 has three binding domains: a high-affinity binding domain, a glycosaminoglycan-binding domain [15], and another high-affinity binding domain located in the N-terminus of the cytokine [16,17]. Previously, Fervert, et al., showed that the IL-8 glycosaminoglycan binding domain determines the location where IL-8 binds in lung tissue, a process mediated by heparan sulphate or chondroitin sulphate [18].
Supplementary material 3. Three oligosaccharyltransferase (O-OTase) mutants (∆pglL, ∆pglC and a double mutant) of the MC58 strain were generated and examined for binding to IL-8 and TNF-α. All three mutants exhibited significant reductions in binding to both IL-8 and TNF-α (S3-Fig. and Fig. 1c main paper). In contrast, a ∆lgtF mutant of the MC58 strain (defective for the synthesis of the polysialic acid capsule or the lipooligosaccharide; LOS), did not exhibit any defects in bacterial binding to IL-8 (S3-Fig.) [19].
S3-Figure. The binding of IL-8 to N. meningitidis is glycan mediated.
Plates were coated with IL-8 and probed with DIG labeled bacteria. Strains that were deficient in O-glycotransferase (ΔpglC) exhibit less IL-8 binding. However, the binding of IL-8 to the ΔlgtF mutant was comparable to the WT strain. In addition, the ΔpilQ and ΔpilE mutants
were used for assessment. The values are representative of three independent experiments (p<0.0001, one-way ANOVA).
Supplementary material 4. Recombinant PilQ binds to human cytokines. To confirm PilQ specificity in binding to cytokines, the binding of purified recombinant PilQ (which is unlikely to be glycosylated in the same fashion as endogenous meningococcal PilQ), to cytokines was compared to purified recombinant PorA (an outer membrane protein). ELISA plates were coated with IL-8 and TNF-α and probed with either purified recombinant PorA or PilQ. The results revealed that recombinant PilQ binds significantly to IL-8 and TNF-α but only marginally to recombinant PorA (p< 0.0001, t-test).
S4-Figure. The purified recombinant PilQ was used for binding to cytokines in comparison to purified recombinant PorA. Only PilQ exhibited strong binding whilst PorA did not, suggesting a protein-protein interaction.
The results shown are expressed as means ± SEM for three independent experiments
carried out in triplicate. The asterisks indicate P values of 0.05, t-test.
Supplementary material 5. Characterization of purified PilQ for possible glycosylation. It has been previously shown that the PilQ subunit of Type IV pili plays an important role in the interaction between cytokines and bacteria. PilQ was purified from WT strain MC58 and analyzed by mass spectrometry (specifically, a nanoflow LC system coupled to a Orbitrap Velos). The sample was reduced and alkylated prior to digestion as previously described [20]. After digestion, the samples were subjected to LC-MS/MS analysis using a hybrid linear ion trap-Orbitrap Velos mass spectrometer that was operated in data-dependent mode and automatically switched to MS/MS mode. MS-spectra were acquired in the Orbitrap, while MS/MS-spectra were acquired in the LTQ-trap. For each MS scan, the ten most intense, double, triple and quadruple-charged ions were sequentially fragmented in the linear trap by collision-induced dissociation. All tandem mass spectra were searched by Proteome Discoverer (Thermo Scientific, San Jose, CA, USA), incorporating MASCOT (Matrix Science, London, UK) against the SwissProt database (released 2011-06). The coverage of the analyzed peptides was 76% of the PilQ protein sequence, and none of the peptides indicated any post-translational modification by glycan insertion. All MS/MS spectra were also manually searched for diagnostic glycan ions without any match.
MNTKLTKIIS GLFVATAAFQ TASAGNITDI KVSSLPNKQK IVKVSFDKEI VNPTGFVTSS PARIALDFEQ TGISMDQQVL EYADPLLSKI SAAQNSSRAR
LVLNLNKPGQ YNTEVRGNKV WIFINESDDT VSAPARPAVK AAPAAPAKQQ AAAPSTKSAV SVSEPFTPAK QQAAAPFTES VVSVSAPFSP AKQQAAASAK
QQAAAPAKQQ AAAPAKQQAA APAKQTNIDF RKDGKNAGII ELAALGFAGQ PDISQQHDHI IVTLKNHTLP TTLQRSLDVA DFKTPVQKVT LKRLNNDTQL
IITTAGNWEL VNKSAAPGYF TFQVLPKKQN LESGGVNNAP KTFTGRKISL DFQDVEIRTI LQILAKESGM NIVASDSVNG KMTLSLKDVP WDQALDLVMQ
ARNLDMRQQG NIVNIAPRDE LLAKDKALLQ AEKDIADLGA LYSQNFQLKY KNVEEFRSIL RLDNADTTGN RNTLISGRGS VLIDPATNTL IVTDTRSVIE
KFRKLIDELD VPAQQVMIEA RIVEAADGFS RDLGVKFGAT GKKKLKNDTS AFGWGVNSGF GGDDKWGAET KINLPITAAA NSISLVRAIS SGALNLELSA
SESLSKTKTL ANPRVLTQNR KEAKIESGYE IPFTVTSIAN GGSSTNTELK KAVLGLTVTP NITPDGQIIM TVKINKDSPA QCASGNQTIL CISTKNLNTQ
AMVENGGTLI VGGIYEEDNG NTLTKVPLLG DIPVIGNLFK TRGKKTDRRE LLIFITPRIM GTAGNSLRY.
S5-Figure. Green: high confidence; Yellow: moderate confidence; Red: low confidence.
Supplementary material 6: RNA Integrity Assessment
Nm strain MC58 was cultured in D-MEM medium at 40 ng/ml cytokines and samples were taken at different time points from 0 to 24 h of co-culture. Following putative induction of bacterial gene regulation with IL-8 or TNF-α, RNA samples were collected and purified from bacteria grown at different time points. The RNA Integrity Number (RIN) data (S6-Fig. a) indicated that
S6-Figure. Determination of RNA integrity. (a) Bacteria were cultured with or without TNF-α or IL-8 (40 ng/ml). RNA was then extracted at the indicated time points, and RNA integrity was measured using a Bio-Analyzer 2100 (Agilent). (b) Analysis of RNA integrity of deep sequencing samples. RNA integrity number (RIN) values are indicated on the graphs.
RNA quality decreased with prolonged incubation time and revealed that the optimal time point was 9 h (RIN˃9). Following incubation, bacterial RNA was isolated and purified. RNA purity and quality were measured using a Nanodrop 1000 Spectrophotometer and an Agilent 2100 Bioanalyser to obtain an RNA Integrity Number (RIN). The RIN data indicated that RNA quality decreased with prolonged bacterial culture times (S6-Fig. b). All samples (except those incubated for 24 h) presented RNA of a quality that was deemed acceptable (RIN ≥ 7).
Supplementary material 7: Deep sequencing.
Our results indicate that human cytokines (i.e. TNF-α and IL-8) may act either as specificity modulators of the RNA polymerase (RNAP) holoenzyme endowing an ability to recognize different promoters for differential gene expression [21] or as de novo transcription factors. Transcription factors can either promote or repress gene expression, depending on the promoters with which they interact. Nm has a relatively small repertoire of transcription factors [22] compared to E. coli, which encodes 314 transcription factors (of which 35% are activators, 43% are repressors and 22% are dual regulators) [23]. However, Nm is equipped with a global regulator gene named NMB0573 (annotated AsnC), a member of the Lrp-AsnC family of regulators that are widely expressed in both bacteria and archaea. AsnC controls responses to nutrient availability through indicators of general amino acid abundance: leucine and methionine [22]. Deep sequencing results revealed that this gene is up-regulated in TNF-α-induced Nm (see separate supplementary Excel file), indicating that TNF-α can modulate the expression of global regulatory genes and indirectly control genome-wide differential gene expression.