AmiA and AliA peptide ligands, found in Klebsiella pneumoniae, are imported into pneumococci and alter the transcriptome

Abstract

Klebsiella pneumoniae releases the peptides AKTIKITQTR and FNEMQPIVDRQ, which bind the pneumococcal proteins AmiA and AliA respectively, two substrate-binding proteins of the ABC transporter Ami-AliA/AliB oligopeptide permease. Exposure to these peptides alters pneumococcal phenotypes such as growth. Using a mutant in which a permease domain of the transporter was disrupted, by growth analysis and epifluorescence microscopy, we confirmed peptide uptake via the Ami permease and intracellular location in the pneumococcus. By RNA-sequencing we found that the peptides modulated expression of genes involved in metabolism, as pathways affected were mostly associated with energy or synthesis and transport of amino acids. Both peptides downregulated expression of genes involved in branched-chain amino acid metabolism and the Ami permease; and upregulated fatty acid biosynthesis genes but differed in their regulation of genes involved in purine and pyrimidine biosynthesis. The transcriptomic changes are consistent with growth suppression by peptide treatment. The peptides inhibited growth of pneumococcal isolates of serotypes 3, 8, 9N, 12F and 19A, currently prevalent in Switzerland, and caused no detectable toxic effect to primary human airway epithelial cells. We conclude that pneumococci take up K. pneumoniae peptides from the environment via binding and transport through the Ami permease. This changes gene expression resulting in altered phenotypes, particularly reduced growth.

Figure 1

Growth suppression requires uptake of AmiA and AliA ligands via the Ami permease. The S. pneumoniae D39 parent strain was inhibited by AmiA ligand (AKTIKITQTR) and AliA ligand (FNEMQPIVDRQ), but inhibition was greatly reduced in the ∆amiC mutant. This indicates that the peptides are taken up via the Ami permease to act. Nonspecific ligand control, FNEMQPIVDRQAAKG, had little effect on growth of D39 parent strain or the ∆amiC mutant. Growth curves were performed in peptide-free chemically defined medium (CDM) by measuring optical density OD (450 nm) over time in absence or presence of peptide (0.5 mg/ml). Results represent 3 independent experiments, error bars indicate SEM.

Figure 2

AmiA and AliA peptide ligands are taken up via Ami permease. Peptides located inside of S. pneumoniae D39 parent strain (a), but not ∆amiC mutant (b). Representative images of pneumococcal cells after incubation with FITC-labelled AmiA and AliA peptide ligands taken at mid-bacterium localization in z position showing FITC and Brightfield (BF) channel. Scale bar indicates 2 µm for all pictures in a and b.

Figure 3

AmiA and AliA peptide ligands locate inside pneumococcal cells of strain D39. Representative images of S. pneumoniae strain D39 parental strain after 5 min incubation with FITC-labelled AmiA and AliA peptide ligands. Scale bar indicates 2 µm for all pictures treated with the same peptide. Orthogonal view from z stack shows colocalization of peptide in FITC channel with cytosol of bacterium in Brightfield (BF) channel.

Figure 4

AmiA and AliA peptide ligands cause differential gene expression in S. pneumoniae strain D39. AmiA and AliA peptide ligands caused similar changes (upregulation or downregulation) in clusters 1,2,5 and 6, whereas AmiA peptide ligand upregulated and AliA peptide ligand downregulated more genes in cluster 3. Patterns are also different in regions of cluster 4.

Figure 5

Top downregulated and upregulated genes by AmiA peptide ligand. Gene association network of significantly (p value < 0.05) downregulated (log2FC ≤ -2) and upregulated (log2FC ≥ 2) genes in S. pneumoniae strain D39 after 15 min of incubation with AmiA peptide ligand. Lines (edges) represent interactions between 2 genes (nodes) and were imported from the STRING database. Nodes are colour coded according to log2FC expression change: blue represents negative log2FC (downregulated) and red positive log2FC (upregulated). Gene Ontologies or keywords of groups of genes are highlighted in the background.

Figure 6

Top downregulated and upregulated genes by AliA peptide ligand. Gene association network of significantly (p value < 0.05) downregulated (log2FC ≤ -1.25) and upregulated (log2FC ≥ 1.25) genes in S. pneumoniae strain D39 after 15 min of incubation with AliA peptide ligand. Lines (edges) represent interactions between 2 genes (nodes) and were imported from the STRING database. Nodes are colour coded according to log2FC expression change: blue represents negative log2FC (downregulated) and red positive log2FC (upregulated). Gene Ontologies or keywords of groups of genes are highlighted in the background.

Figure 7

Growth curves of Swiss clinical isolates of S. pneumoniae in presence and absence of AmiA and AliA peptide ligands. Effect of peptides on growth was tested on isolates of different serotype (ser) with isolate number in brackets: 3 (1189.65), 8 (1188.31), 9N (1186.70), 12F (1193.68), 19A (1189.53). Growth curves were performed in peptide-free chemically defined medium (CDM) by measuring optical density OD (450 nm) over time in absence or presence of AmiA or AliA peptide ligands (0.5 mg/ml). Results represent 3 independent experiments, error bars indicate SEM.