ABSTRACT Treatment of pneumococcal infections is limited by antibiotic resistance and exacerbation of disease by bacterial lysis releasing pneumolysin toxin and other inflammatory factors.

We identified a previously uncharacterized peptide in the _Klebsiella pneumoniae_ secretome, which enters _Streptococcus pneumoniae_ via its AmiA-AliA/AliB permease. Subsequent

downregulation of genes for amino acid biosynthesis and peptide uptake was associated with reduction of pneumococcal growth in defined medium and human cerebrospinal fluid, irregular cell

shape, decreased chain length and decreased genetic transformation. The bacteriostatic effect was specific to _S. pneumoniae_ and _Streptococcus pseudopneumoniae_ with no effect on

diseases suppressing growth of multiple clinical isolates, including antibiotic resistant strains, while avoiding bacterial lysis and dysbiosis. SIMILAR CONTENT BEING VIEWED BY OTHERS AMIA

AND ALIA PEPTIDE LIGANDS, FOUND IN _KLEBSIELLA PNEUMONIAE_, ARE IMPORTED INTO PNEUMOCOCCI AND ALTER THE TRANSCRIPTOME Article Open access 30 May 2024 AMIA AND ALIA PEPTIDE LIGANDS ARE

SECRETED BY _KLEBSIELLA PNEUMONIAE_ AND INHIBIT GROWTH OF _STREPTOCOCCUS PNEUMONIAE_ Article Open access 23 December 2022 PNEUMOCOCCAL SURFACE PROTEIN A (PSPA) PREVENTS KILLING OF

_STREPTOCOCCUS PNEUMONIAE_ BY INDOLICIDIN Article Open access 09 October 2024 INTRODUCTION Antibiotic resistance is one of the biggest threats to society, with the current trajectory

predicted to lead to 10 million deaths due to antibiotic resistance per year by 20501. The economic impact is also significant, with the cost of antibiotic failure estimated to be €1.5

billion in Europe annually2. Amongst the priority pathogens listed by the World Health Organization is penicillin-non-susceptible _Streptococcus pneumoniae_ (pneumococcus)3,4. These

Gram-positive bacteria are responsible for diseases including meningitis, pneumonia, and septicaemia with significant morbidity and mortality, particularly in young children, the elderly,

and the immunocompromised5. The pneumococcus is the most common cause of community-acquired pneumonia globally5, with antibiotic resistance a growing concern6 and so new treatment strategies

are needed. Prior to the potential invasion, _S. pneumoniae_ must colonize the human nasopharyngeal niche alongside other members of the microbiota. Some commensal microbiota species such

as _Dolosigranulum pigrum_, _Corynebacterium_7,8,9, _Streptococcus salivarius_10, and _Prevotella melaninogenica_11 protect from colonization by _S. pneumoniae_, for example, by modulating

the innate immune response or by l-lactic acid production. _S. pneumoniae_ is also proposed to sense the environment via its transporter Ami-AliA/AliB permease, which takes up oligopeptides,

resulting in gene expression modulation12 and affecting nasopharyngeal colonization13. We previously expressed this transporter’s oligopeptide binding proteins AmiA, AliA, and AliB as

recombinant proteins and identified their peptide ligands from the nasal wash of children. Depending on the peptide, they affected phenotypes such as growth14,15. Some of the peptides had

sequences that matched ribosomal proteins of _Klebsiella pneumoniae_, another resident of the respiratory tract with the potential to cause pneumonia, and were found in the secretome of _K.

pneumoniae_16. It is unknown whether a functional Ami-AliA/AliB permease is required for the effect of such _K. pneumoniae_ peptides, whether peptide uptake is necessary, and whether other

_K. pneumoniae_ secretome peptides that hijack the permease remain to be discovered. To address this, we harvested the extracellular peptidome of actively growing _K. pneumoniae_ in a

peptide-free medium and analysed it by mass spectrometry. We identified a previously uncharacterized peptide of 11 amino acid length, which inhibited the growth of a wide range of clinical

pneumococcal isolates, including those resistant to antibiotics, affected cell shape, decreased chain length, and transformation efficiency. We tested its effects not only in a culture

medium but also in human cerebrospinal fluid (hCSF), at the air interface of primary human airway epithelial cell cultures and in vivo in the rat nasopharynx in the presence of a microbiota.

Following uptake via the Ami permease, the pneumococcal transcriptome and proteome were altered, and pneumococcal colonization decreased. We propose that this interspecies peptide has

therapeutic potential to treat pneumococcal diseases. RESULTS _KLEBSIELLA PNEUMONIAE_ RELEASES A PREVIOUSLY UNCHARACTERIZED RIBOSOMAL PEPTIDE We extracted peptides secreted by live _K.

pneumoniae_ into a chemically defined medium (CDM) by performing solid-phase extraction and identified the peptides by LC–MS/MS. Along with previously identified AmiA and AliA peptide

ligands16, we found another ribosomal peptide of similar amino acid length. Three peptides were present, which included the sequence VNATDEDRWNA, found in ribosomal protein S14: 11-amino

acid VNATDEDRWNA, 13-amino acid SDVNATDEDRWNA, and 15-amino acid IISDVNATDEDRWNA. The 11-amino acid peptide is abbreviated here as V11A. PEPTIDE V11A SUPPRESSES PNEUMOCOCCAL GROWTH We tested

the effect of peptide V11A and the 13- and 15-amino acid versions on the growth of _S. pneumoniae_ laboratory strain D39 in CDM. V11A, but not the longer peptides, suppressed growth in a

dose-dependent manner (Fig. 1a). By performing BLAST with the sequence of peptide V11A, we found amino acid differences at positions 3, 4, 7, and/or 10 in various species. For example,

serine, instead of threonine, at position 4 was found in _Escherichia coli, Enterobacter hormaechei_, and _Haemophilus parainfluenzae_ strains. We therefore tested whether these differences

influence the growth-inhibiting effect of peptide V11A on _S. pneumoniae_. We found that peptides with substitutions at positions 4 or 10 did not suppress pneumococcal growth, whereas

substitutions at positions 3 or 7 had no effect (Fig. 1b). Since peptide V11A inhibited the growth of _S. pneumoniae_ laboratory strain D39 (Fig. 1a) we tested its effect on diverse clinical

isolates. We found that peptide V11A inhibited the growth of a wide range of isolates, regardless of serotype or antibiotic resistance (Table 1 and Supplementary Fig. 1). In contrast to

previously identified _K. pneumoniae_ ribosomal peptides, AmiA ligand (AKTIKITQTR) and AliA ligand (FNEMQPIVDRQ), V11A suppressed the growth of a trimethoprim-sulfamethoxazole resistant

isolate 1154.75 (serotype 23F) (Fig. 1c). Growth of this isolate (1154.75) was also suppressed by V11A in human cerebrospinal fluid (hCSF) from five different donors, mimicking the

environment of _S. pneumoniae_ during pneumococcal meningitis (Fig. 1d, Supplementary Fig. 2). To test the specificity of growth inhibition by peptide V11A, we tested its effect on growth of

other common bacterial colonizers of the nasopharynx. None were inhibited by V11A, except _Streptococcus pseudopneumoniae_, a very close relative of _S. pneumoniae_ (Table 1 and

Supplementary Fig. 1). There were two pneumococcal strains that were not inhibited by V11A: South African strain 17619 and laboratory strain R6, which we have shown previously both have

mutated AmiA and whose growth is also not suppressed by the AmiA and AliA peptides16. A BLAST search of the AmiA sequence covering the relevant mutation was performed to determine its

frequency: YGYVYTADPETLDYLISRK (mutation in bold and underlined). Of the 5000 sequences examined, only 4 (0.08%) had this mutation. A time-kill assay showed that V11A had a bacteriostatic

effect on pneumococcus in contrast to the bactericidal antibiotic penicillin G, used as a control (Fig. 1e). In summary, peptide V11A suppressed growth of a range of pneumococcal isolates in

CDM and suppressed growth of a clinical pneumococcal isolate in hCSF, the effect was bacteriostatic, species-specific and the peptide length and sequence were critical. PNEUMOCOCCAL GROWTH

INHIBITION BY V11A REQUIRES UPTAKE VIA AMI-ALIA/ALIB PERMEASE As peptide V11A did not inhibit two pneumococcal strains with mutated AmiA we hypothesized that the Ami-AliA/AliB oligopeptide

permease is involved in the response to V11A. We confirmed this with a mutant in which AmiC, one of the two permease subunits, was disrupted. In the Δ_amiC_ mutant the growth suppression

effect of V11A was lost (Fig. 2a). To determine whether peptide V11A is taken up into _S. pneumoniae_ D39 and whether a functional Ami-AliA/AliB oligopeptide permease is necessary for this,

we incubated D39 and the Δ_amiC_ mutant for 5 min with FITC-labelled peptide V11A and performed epifluorescence microscopy. We found clear homogeneous FITC staining intracellularly only in

the D39 strain with a functional permease and greatly reduced uptake in the Δ_amiC_ mutant (Fig. 2b). In the orthogonal view of a z-stack, we see the FITC staining inside the bacterial cell

and not on the membrane (Supplementary Fig. 3). For versions of peptide V11A with amino acid differences that did not suppress pneumococcal growth (positions 4 or 10, Fig. 1b), we found

greatly reduced uptake of FITC-labelled peptide in the cytoplasm of either D39 or its ∆_amiC_ mutant (Supplementary Fig. 4). In conclusion, we found that pneumococcal growth suppression by

peptide V11A requires uptake of the peptide via a functional Ami-AliA/AliB oligopeptide permease. PEPTIDE V11A AFFECTS PNEUMOCOCCAL MORPHOLOGY, CHAIN LENGTH AND TRANSFORMATION The antibiotic

aztreonam and β-lactamase inhibitor clavulanic acid have been shown to increase pneumococcal chain length and also induce competence17, which can allow the pneumococcus to take up and

incorporate exogenous DNA, supporting the rapid spread of antimicrobial resistance. We, therefore, analysed the effect of peptide V11A on pneumococcal cell morphology, chain length and

transformation rate. We grew three different pneumococcal strains with or without peptide V11A until the mid-log phase and performed microscopy at 40x and 100x magnification. Untreated

bacteria within the same chain had homogenous size and shape. In the V11A peptide-treated samples, bacteria within the same chain had heterogenous shapes and sizes, with some cells enlarged

and rounder than in the untreated sample (Fig. 3a). For all three pneumococcal strains, peptide V11A also significantly decreased chain length (Fig. 3b, Supplementary Fig. 5). We tested the

effect of peptide V11A on competence-stimulating peptide (CSP-1)-mediated transformation rate in strain D39. V11A, but not the control peptide (V11A with an amino acid difference at position

4), reduced the mean transformation rate from 1.44% to 0.169%, which equals an 8.5-fold reduction (Fig. 3c). In summary, we found that V11A changes pneumococcal cell morphology, decreases

the chain length and transformation rate. PEPTIDE V11A DOWNREGULATES GENES INVOLVED IN AMINO ACID AND PROTEIN METABOLISM To decipher the mechanism by which V11A caused the phenotypic changes

described above, we identified differentially expressed genes in pneumococcal strain D39 treated with peptide V11A compared to the untreated control. Upon V11A treatment, more genes were

downregulated than upregulated, as seen in the volcano plot in Supplementary Fig. 6a. Eighty-nine genes underwent a significant change in expression equal or greater than 1.25 log2 fold

change (log2FC), of which 64 were downregulated and 25 were upregulated. With these down- and upregulated genes, we performed Gene Ontology (GO) enrichment analysis. For upregulated genes,

we did not find biological processes with statistically significant enrichment compared to the genome background. We found several biological processes enriched in the downregulated genes,

mainly amino acid biosynthesis, import and transport, but also peptide and protein transport (Supplementary Fig. 6b). With the same cutoffs, we made a gene interaction network (Fig. 4). We

see that downregulated genes cluster to the following: AmiA permease (_ami_ genes and _aliA_), branched-chain amino acid biosynthesis (_ilv_ genes), branched-chain amino acid transporter

(_liv_ genes) and riboflavin synthesis (_rib_ genes). One interesting gene cluster of upregulated genes is fatty acid biosynthesis (_fab_ genes). At the level of single genes, the most

downregulated gene was SPD_0161, a Mn2+/Fe2+ transporter and the most upregulated gene was SPD_1524 (transcriptional regulator, _gntR_). We confirmed significant downregulation of _aliA,

ilvB_, _livJ_ and significant upregulation of _gntR_ by real-time RT-PCR (Supplementary Fig. 6c). We found _codY_, a global regulator of protein metabolism, downregulated −1.2 log2FC. We did

not find any _com_ genes differentially expressed, however, we found downregulation of other genes potentially involved in competence18,19,20: _ssb_ with −0.5 log2FC, _hrcA_ with −1.1

log2FC and _grpE_ with −1.2 log2FC. We found _dnaJ_ and _dnaK_ downregulated with −1.6 log2FC and −1.3 log2FC, respectively. _Lic_ genes (_licC, licD1-D3, licT_) were downregulated by −0.3

to −0.7 log2FC, _phtD_, _phtE_ downregulated −1.1 and −1.9 log2FC, respectively. _CbpA_ was downregulated −0.5 log2FC and _nanA _-1 log2FC. To summarize, we found that V11A altered the

pneumococcal transcriptome, mainly downregulating genes involved in amino acid and protein metabolism, but also transcriptional regulators. PEPTIDE V11A ALTERS THE PNEUMOCOCCAL PROTEOME We

also analysed the effect of peptide V11A on the proteome of _S. pneumoniae_ strain D39. We identified some proteins only detected in the sample without peptide treatment as “off” and

proteins only detected in the peptide treatment sample as “on” in Table 2 for _S. pneumoniae_ strain D39 and in Supplementary Tables 1a and 1b for strains 106.66 and 208.41. We found

choline-binding protein D to be switched off by V11A treatment and dehydratase FabZ to be turned on in strain D39. In strain 106.66, we found the permease protein of the branched-chain amino

acid ABC transporter (encoded by the _livH_ gene) and riboflavin biosynthesis protein RibBA to be turned off. Thus, we found alteration of the proteome by V11A with some proteins turned on

or off in line with the transcriptomic changes. NO TOXIC EFFECT WAS DETECTED FOR PEPTIDE V11A TO HUMAN AIRWAY EPITHELIAL CELL CULTURES OR ZEBRAFISH LARVAE Well-differentiated airway

epithelial cell (AEC) cultures are organotypic cell cultures with air–liquid interfaces and recapitulate many aspects of the respiratory tract, including airway cell heterogeneity, mucus

production and ciliary beating activity21. To evaluate whether peptide V11A is cytotoxic to primary human AEC (hAEC) cultures, we incubated the hAEC cultures together with peptide V11A for

30 h. We collected the medium from the apical and basolateral side and determined cytotoxicity calculated as %LDH release using the formula of Rayamajhi et al.22. The LDH release was the

same and low in media collected from hAEC cultures treated with and without peptide V11A. Therefore no cytotoxicity was detected (Supplementary Fig. 7a). Immunofluorescence staining also

shows the same cell morphology with intact tight junctions and ciliated cells present in all samples (Supplementary Fig. 7b). To test whether the peptide is toxic in vivo, zebrafish larvae

were exposed to 0.5 mg/ml or 0.25 mg/ml V11A and their swimming behaviour determined in light and dark conditions. This light–dark locomotion test is a well-established metric for toxicity

measurements that can reveal, for example, reactivity and muscular weakness23,24. The presence of the peptide did not affect the swimming behaviour (Supplementary Fig. 7c). In conclusion, no

toxic effect of peptide V11A was detected in vitro to hAEC cultures or in vivo to zebrafish larvae. PEPTIDE V11A INHIBITS PNEUMOCOCCAL ADHERENCE TO HUMAN AIRWAY EPITHELIAL CELL (HAEC)

CULTURES AND COLONIZATION OF THE RAT NASOPHARYNX Increased chain length has been associated with increased adherence of pneumococci to epithelial cells25. As peptide V11A decreased

pneumococcal chain length (Fig. 3b, Supplementary Fig. 5) and was not toxic to hAEC cultures (Supplementary Fig. 7), we tested its effect on pneumococcal adherence in vitro using the same

model. We exposed hAEC cultures from three different donors to _S. pneumoniae_ strain D39 and quantified the number of adhered bacteria on the apical surface after removing non-adhered

bacteria 24 h post-inoculation. Peptide V11A, but not the control peptide (V11A with an amino acid difference at position 10), decreased bacterial adherence to 22% of that of the control

without peptide (Fig. 5a). The control peptide had no effect on adherence. To test whether peptide V11A had an effect in vivo in the presence of the microbiota, we inoculated rat pups

intranasally with _S. pneumoniae_ strain 1154.75 (serotype 23F) with or without peptide V11A and quantified bacterial load in nasal wash and nasopharynx 24 and 48 h later. At 24 h,

pneumococci were detected in the nasopharynx of 9 out of 11 rats (81.81%) in the absence of V11A and in 3 out of 11 (27.27%) in the nasopharynx of rats that had received V11A (Chi-squared

test: _p_ = 0.03228). Bacterial loads at 24 h are shown in Fig. 5b and were significantly lower in the nasopharynx of rats that had received V11A (_p_ = 0.01367). Fewer bacteria in the

nasopharynx of the peptide-treated rats than controls were also seen at 48 h (not significant, _p_ = 0.30, Supplementary Fig. 8a). Bacterial load in the nasal wash was also reduced

significantly in presence of V11A at 24 h (_p_ = 0.01989) and 48 h (_p_ = 0.01704) (Supplementary Fig. 8b). Therefore, peptide V11A inhibited pneumococcal adherence in vitro to hAEC cultures

and colonization in vivo of the rat nasopharynx. DISCUSSION We found a previously uncharacterized peptide, V11A, in the secretome of _K. pneumoniae_, which modulates pneumococcal

phenotypes, particularly growth and colonization. For the first time we show that an interspecies peptide suppresses pneumococcal growth not only in a defined medium but in human

cerebrospinal fluid, a site of infection in pneumococcal meningitis. Growth suppression is achieved following uptake via the Ami-AliA/AliB permease leading to transcriptional and proteomic

changes. V11A inhibited pneumococcal growth in a dose-dependent manner, with the effect lost for versions of the peptide longer than 11 amino acids or with differences at positions 4 or 10.

These changes presumably prevent the peptide from binding to its target on the pneumococcus. V11A suppressed the growth of genetically diverse clinical isolates of pneumococcus, which bodes

well for its use as a therapeutic agent. Two strains unaffected by V11A are known to have mutations in the protein AmiA which led us to speculate that AmiA is required for the effect of

V11A16. The role of the Ami-AliA/AliB oligopeptide permease in the uptake of V11A was confirmed by the observation that deletion of one of the permease subunits, AmiC, abrogated the effect

of the peptide on growth and that fluorescently labelled V11A was taken up by the D39 parent strain, but not the ∆_amiC_ mutant. V11A did not affect the growth of other bacterial colonizers

of the nasopharynx tested: _S. mitis, H. influenzae, S. aureus_ or _K. pneumoniae_. Another proteinaceous interspecies molecule from the genus _Lysinibacillus_ was found previously, which

depends on the Ami permease for growth inhibition of not only _S. pneumoniae_ but also _S. mitis_26. V11A therefore appears to have a more species-specific effect. This specificity towards

pneumococci is an attractive feature and an advantage over antibiotics which affect multiple species and may lead to dysbiosis of the microbiota. This is also an advantage of V11A over

antimicrobial peptides (AMPs) of the host innate immune response, which tend to have a broad effect27. In contrast to β-lactam lytic antibiotics, such as penicillin, used to treat

pneumococcal infections, V11A had a bacteriostatic effect. By reducing the number of bacteria but not causing their lysis, little pneumolysin should be released compared to treatment with a

β-lactam antibiotic. This would be a significant advantage in treatment, particularly of meningitis where antibiotic-induced bacterial lysis causes the release of pneumolysin, which

exacerbates the inflammatory response, neuronal death, and therefore disease28,29. The host cytokine response to bacterial lysis can reduce the chance of survival and increase neurological

sequelae such as hearing loss and learning difficulties in patients who do survive30. We can associate many of the phenotype changes observed with changes in the transcriptome or proteome

caused by peptide V11A. The genes most downregulated by peptide V11A were all associated with transport, energy or the synthesis and transport of branched-chain amino acids. Efficient

acquisition of branched-chain amino acids is associated with nasopharyngeal colonization31. The top-upregulated gene was _gntR_, which has a role in sensing environmental and nutritional

cues32. The proteins FabK, FabG and FabF, which are encoded in the _fabKDGF-accB-fabZ-accCDA_ operon, participate in enzymatic reactions of fatty acid biosynthesis by adding

malonyl-coenzyme-A molecules to the growing hydrocarbon chain33. We found FabZ protein expression switched on in the presence of peptide V11A along with heterogeneous cell sizes and

decreased chain length. _Fab_ and _acc_ genes were also upregulated in the transcriptome following peptide exposure. Another study found that a competence-associated quorum peptide, BriC,

altered fatty acid biosynthesis in _S. pneumoniae_ and also promoted nasopharyngeal colonization34. We found modulation of fatty acid biosynthesis by interspecies peptide V11A. This,

together with observed phenotype changes, is in agreement with a study using a CRISPRi screen where repression of _fabD_, _fabK, fabZ, accB_ and _accD_ led to irregular cell shapes and

longer chains35. Cell chaining is associated with a longer competence and transformation time window as cell chains retain, rather than diffuse the CSP peptide17. This is compatible with our

finding that in addition to decreased chain length, we also found that peptide V11A decreased the transformation efficiency of _S. pneumoniae_. Competence contributes to the astonishing

genomic plasticity of _S. pneumoniae_, which can facilitate antigenic variation, vaccine escape and the acquisition of antibiotic resistance19. For a potential therapeutic agent, decreasing

the ability of the pneumococcus to acquire antibiotic resistance is of advantage. We did not find any _com_ genes, which are involved in regulating and developing competence for genetic

transformation36, differentially expressed upon peptide V11A treatment to explain the reduction of transformation efficiency. However, we found downregulation of other genes, namely _ssb_,

_hrcA_, _grpE_, _dnaK_ and _dnaJ_, which have been associated with regulating competence18,20. The _ssbB_ gene is regulated by the alternative sigma factor ComX18 and therefore expressed

uniquely during competence for genetic transformation. It encodes an alternative single-stranded DNA-binding protein that might increase the likelihood of multiple transformation events in

the same cell37. The pneumococcal competence response exhibits a broad phenotype, combining chaperone and protease production with genetic recombination, which includes induction of such

stress response proteases and chaperones38. Furthermore, in the proteomics data, CbpD was turned off by V11A peptide treatment. CbpD is a competence-specific murein hydrolase that lyses a

subfraction of the pneumococcal population or close relatives in order to release DNA for uptake39,40 and is produced exclusively by competent cells41. One study found that deletion of CbpD

reduced transformation efficiency in _Streptococcus thermophilus_42. The same has been observed in _Streptococcus sanguinis_ lacking LytF43 and _Streptococcus suis_, lacking CrfP44, which

are both functional analogues of CbpD. The CodY regulon is involved in the regulation of genes involved in amino acid metabolism, carbon metabolism and iron uptake and among genes described

as differentially expressed in D39∆_codY_ were also _aliA, ilv_ and _liv_ genes45. In our transcriptomics data _aliA, ilv_ and _liv_ genes and _codY_ are differentially expressed upon

peptide V11A treatment. Colonization, a prerequisite for nasopharyngeal carriage of _S. pneumoniae_, allows pneumococcus to spread between hosts and to cause invasive disease. Adherence to

host cells is one of the main features facilitating colonization46. The Ami permease has been suggested previously to modulate pneumococcal adherence to epithelial cells by modulating the

expression of adhesins on the pneumococcal surface during the first stages of colonization47. Increased bacterial chain length has been associated with enhanced adherence of pneumococci to

epithelial cells25. In accordance with this, peptide V11A, which decreased chain length, inhibited pneumococcal adherence to human airway epithelial cell cultures. V11A downregulated _phtD_,

_phtE_, c_bpA_ and _nanA_. Pneumococcal surface and histidine triad proteins PhtD and PhtE play a role in adherence to respiratory mucosa and anti-PhtD antibodies can inhibit bacterial

attachment48,49. CbpA binds the host proteins factor H and vitronectin46, which might facilitate adherence to host cells and NanA has been suggested to function as an adhesin50. Furthermore,

V11A downregulated _lic_ genes, including _licC_, _licD1-D3_, _licT_. LicD2 mutants showed decreased transformation competence, decreased adherence, reduced nasopharyngeal colonization and

reduced virulence in another study51. To have potential as a therapeutic, the peptide must have an effect in vivo. We found that V11A significantly reduced the percentage of rats colonized

and bacterial load in the nasopharynx and nasal wash of infant rats, indicating its effectiveness in a nutritionally complex environment in the presence of a microbiota. V11A also reduced

the growth of isolate 1154.75 (serotype 23F), which the AmiA and AliA ligands did not. Furthermore, AmiA and AliA peptides increased the chain length of bacteria and did not affect adherence

to epithelial cells15, while V11A decreased both chain length and adherence. While the AmiA and AliA peptides reduced the transformation rate by 3-fold15, V11A caused an 8.5-fold reduction.

The limitations of the study include that we used high doses of peptides, but optimization, for example, of delivery, could reduce effective doses. Future studies would also need to test

whether resistance to peptides develops over time, as happens with antibiotics. Alterations to the genes encoding penicillin-binding proteins, involved in cell wall construction, allow

bacteria to escape the action of the β-lactam antibiotics52,53. However, the peptide acts by specific binding to the substrate-binding protein of an ABC transporter which has a role in

sensing peptides in the environment. Mutation would mean the loss of this function. Therefore, we predict this is less likely to occur. This is supported by the fact that a BLAST search of

AmiA and AliA proteins indicates that they are highly conserved16 and the mutation in AmiA of strain R6, associated with lack of response to V11A, was found in fewer than 0.08% of

pneumococcal genomes. A limitation of the colonization study is that peptide was administered simultaneously with the bacteria, in contrast to the situation of treatment when

colonization/infection would be established then peptide administered. However, the experiment established proof of concept that peptides can be effective in vivo. We have shown that

interspecies peptide V11A produced by _K. pneumoniae_ modulated phenotypes of _S. pneumoniae_, particularly suppressing its growth in the medium and in human cerebrospinal fluid. We

speculate that the import of the peptide via the pneumococcal AmiA-AliA/AliB permease may be advantageous to _K. pneumoniae_ in the shared niche of the nasopharynx. Uptake of peptide V11A

led to the downregulation of genes for amino acid biosynthesis, caused irregular cell shape, decreased chain length and decreased genetic transformation. The bacteriostatic effect was

species-specific and dependet on peptide sequence and length. The peptide reduced pneumococcal adherence to hAEC cultures and colonization of rat nasopharynx, without apparent toxicity. We,

therefore, propose that peptide V11A has potential as a therapeutic agent against pneumococcal diseases with the advantages of effectiveness against antibiotic-resistant strains, avoiding

bacterial lysis and the subsequent damaging inflammatory response and specificity of action, avoiding dysbiosis of the microbiota. METHODS BACTERIAL STRAINS AND CULTURE CONDITIONS Bacterial

strains and culture conditions were used as described previously16. Briefly, the bacterial strains were stored at -80 °C in the Protect Microorganism Preservation System (Technical Service

Consultants Ltd.). As previously described, _S. pneumoniae_ was streaked out on CSBA plates, _H. influenzae_ on CHOC plates and both grown overnight at 37 °C and 5% CO2; _K. pneumoniae_ and

_S. aureus_ were streaked out on CSBA plates and grown overnight at 37 °C and atmospheric CO2 concentration. Swiss clinical _S. pneumoniae_ strains originated from a strain collection

obtained from nationwide surveillance for nasopharyngeal _S. pneumoniae_ isolates from children with respiratory infection54 or were kindly provided by Carlo Casanova, Institute for

Infectious Diseases, Bern. Strains used, including reference strains, are shown in Table 1. The D39 parent strain (ds865, D39 (∆_comAB::erm_)) and its mutant ∆_amiC_ (ds1039, D39,

∆_amiC::Janus_) were used in Fig. 226. Swiss clinical isolates of commensal streptococci _S. pseudopneumoniae_ (410.05) and _S. mitis_ (3308.46) were also used16. As previously, chemically

defined media (CDM)16, BHI medium and PBS were made in-house. IDENTIFICATION OF PEPTIDES IN THE SECRETOME Peptides secreted by _K. pneumoniae_ were identified as described previously by mass

spectrometry16. PEPTIDES Synthetic peptides (Genscript, Thermo Fisher Scientific) were ordered with ≥95% purity. Peptides for fluorescent microscopy were labelled at the C-terminus with

Lys(FITC). For human airway epithelial cell cultures and in vivo experiments (zebrafish larvae, rats), peptides were endotoxin-free and with TFA removal (salt form: acetate). HUMAN

CEREBROSPINAL FLUID Residual human cerebrospinal fluid (hCSF) from five anonymized patients undergoing lumbar puncture in 2023 due to non-inflammatory conditions was used for growth assay.

GROWTH ASSAY Growth assays were done as described previously16 and plotted in R with error bars representing SEM of 3 independent biological replicates. For growth in hCSF, bacteria were

inoculated as described elsewhere55 with twice the inoculum in PBS compared to the growth assay in CDM due to anticipated slower growth in hCSF. TIME-KILL ASSAY Following overnight culture

on agar plates, bacteria were sub-cultured in BHI medium overnight for approximately 7 h until the cultures reached OD600nm = 0.5 in mid-log phase. 500 µl of the overnight culture were

sub-cultured in 5 ml of fresh BHI until OD600nm = 0.5, centrifuged at 3000×_g_ for 7 min and the pellet resuspended in 5 ml CDM. The absorbance was adjusted in CDM to OD600nm = 0.05, then 3 

ml of this bacterial suspension was mixed with 7 ml CDM to reach ca. 5 × 106 cfu (colony forming units)/ml. Penicillin G and peptide were resuspended to 0.5 mg/ml with the bacterial

suspension. In Eppendorf tubes, 1 ml cultures without or with Penicillin G or peptide were grown in a water bath at 37 °C and every 30 min for 5 h, 10 µl were removed to plate out dilutions

onto CSBA plates. Viable bacteria counts were determined after overnight incubation of the CSBA plates at 37 °C and 5% CO2. EPIFLUORESCENCE MICROSCOPY Following overnight culture on agar

plates, _S. pneumoniae_ D39 parent strain and D39∆_amiC_ were grown in BHI to OD600nm = 0.1–0.2, 1 ml culture centrifuged at 10,000×_g_ for 1 min and the pellet resuspended in 100 µl BHI and

50 µl of 1 mg/ml fluorescently labelled peptide. After 5 min incubation time, the bacteria were washed in 1 ml PBS three times. After the last wash, the pellet was resuspended in 40 µl of

PBS and 1.5 µl was spotted onto an acrylamide pad in a gene-frame on a microscope slide. We prepared acrylamide slides by sticking a gene-frame (Thermo Scientific, AB0578) onto a glass slide

and mixing the following in a tube per acrylamide slide: 370 µl PBS, 125 µl 40% acrylamide/bis-acrylamide 29:1 (Sigma-Aldrich, A7802), 0.5 µl TEMED (Sigma-Aldrich, T9281), 5 µl freshly

prepared 10% APS (Sigma-Aldrich, A3678). From this mix, we poured 500 µl into the gene-frame and added another glass slide to close it. After 30 min at room temperature, we gently slid off

one glass slide, cut the acrylamide pads into small pieces and incubated them in PBS for at least 30 min before use. Microscopy was performed using a Zeiss Axio Imager M1 fluorescence

microscope with a ×100 oil immersion objective (EC Plan-Neufluoar 100x/1.30 Oil M27), filter with 493 nm excitation and 517 nm emission wavelength for the FITC channel and images

photographed by a Zeiss Axiocam 712 mono camera. We edited pictures for Fig. 2b with the same settings for all samples to maintain comparability between samples and intensity of fluorescence

using the Software Zeiss ZEN 3.8: denoising the brightfield channel picture with setting real wavelets to 1.0, followed by adjusting the histogram upper white limit in the brightfield

channel to 6000 and in the FITC channel to 5000. BACTERIAL SHAPE CHARACTERIZATION Bacterial culturing was done as described for the growth assay for 2.5 h then 800 µl were centrifuged at

3000×_g_ for 7 min. Most of the supernatant was discarded and the pellet resuspended in the remaining volume of CDM (ca. 30 µl). A bacterial suspension of 10 µl was mixed with 2 µl

fluorescein isothiocyanate (FITC)-Dextran solution (Sigma-Aldrich, resuspended to 10 mg/ml in water). Of this mixture, 10 µl were pipetted onto a microscope slide and the coverslip was

applied firmly. The slides were viewed using a Zeiss Axio Imager M1 fluorescence microscope with a ×100 oil immersion objective and brightfield images photographed by a Zeiss AxioCam HRc

camera. CHAIN FORMATION Bacteria were cultured as described above and 8 µl were pipetted onto a microscope slide, air-dried, heat-fixed by flame and Gram-stained using the Aerospray TB Slide

Stainer (ELITechGroup). Pictures were taken with ×40 objectives in the Cytation 5 multimode reader (Biotek) with the Gen5 Software (Biotek). Microscopy image analysis of chain length was

done using MicrobeJ56. Results for each sample represent measurements of >1200 bacterial chains from at least 12 pictures of 3 independent biological replicates. Bacteria chain detection

settings (=objects) were set to exclude objects on the picture edge, objects with branching and only recognized as objects with a pixel width of 5-15. TRANSFORMATION ASSAY The protocol was

adapted from elsewhere57 to the following: _S. pneumoniae_ strain D39 was grown in BHI + 5% FCS to OD600nm = 0.5, then 150 µl sub-cultured in 9.5 ml of fresh BHI + 5% FCS until OD600nm = 

0.1. After centrifugation at 3000×_g_ for 7 min the pellet was resuspended in the same volume of CDM. One ml of the bacterial suspension with 100 ng/ml CSP-1 (EMRLSKFFRDFILQRKK) with and

without 0.5 mg/ml peptide V11A was incubated for 12 min at 37 °C. After 100 µl were transferred into a new prewarmed tube and 1 µg chromosomal DNA from streptomycin-resistant strain 104.37

was added, and the culture was incubated for 20 min at 30 °C, then 900 µl prewarmed CDM was added, and the culture incubated for 90 min at 37 °C. Serial dilutions in PBS were plated onto

CSBA plates with and without 200 µg/ml streptomycin. The number of colonies was counted after overnight incubation and the transformation rate was calculated. GENE EXPRESSION ANALYSIS BY

RNA-SEQ AND RT-QPCR Following overnight culture on agar plates, _S. pneumoniae_ strain D39 was grown in BHI + 5% FCS to OD600nm = 0.5 and 100 µl of the overnight culture was sub-cultured in

10 ml of fresh CDM until OD600nm = 0.2. The culture was split into two 5 ml cultures and 0.5 mg/ml peptide was added to one sample. After 15 min incubation at 37 °C, transcription was

stopped with RNAprotect bacteria reagent (Qiagen) and total RNA was isolated with the RNeasy kit (Qiagen) according to the manufacturer’s manual. RNA concentration and integrity were

assessed with Nanodrop (Thermo Scientific) and Agilent Biolanalyzer (Agilent) before submitting 6 biological replicates of each sample to the Next Generation Sequencing Platform (University

of Bern), where quality control assessments, rRNA depletion, generation of libraries and sequencing were carried out. At the Next Generation Sequencing Platform (University of Bern), the

quantity and quality of the purified total RNA were assessed using a Qubit 4.0 fluorometer (Thermo Fisher Scientific) with the Qubit RNA BR Assay Kit (Thermo Fisher Scientific, Q10211) and

an Advanced Analytical Fragment Analyser System using a Fragment Analyser RNA Kit (Agilent, DNF-471), respectively. Thereafter, 150 ng of input RNA was depleted of ribosomal RNA using a

RiboCop rRNA Depletion Kit for Gram-Positive Bacteria (G+) (Lexogen, SKU 127) following the Lexogen User guide 125UG246V0102. Next, cDNA libraries were generated using a CORALL RNA-Seq V2

Library Prep Kit with UDI 12 nt Set A3 (Lexogen, SKU 173) according to the RTL protocol and 15 PCR cycles (Lexogen User Guide 171UG394V0100). The resulting cDNA libraries were evaluated

using a Qubit 4.0 fluorometer with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Q32854) and an Agilent Fragment Analyser with an HS NGS Fragment Kit (Agilent, DNF-474),

respectively. Pooled cDNA libraries were sequenced paired-end using a NextSeq 1000/2000 P2 Reagents v3 (200 cycles; Illumina, 20046812) on an Illumina NextSeq 1000 instrument. The run

produced, on average, 26 million reads/library. The quality of the sequencing run was assessed using Illumina Sequencing Analysis Viewer (Illumina version 2.4.7) and all base call files were

demultiplexed and converted into FASTQ files using Illumina bcl2fastq conversion software v2.20. Lexogen (Vienna, Austria) performed quality control, read processing and differential gene

expression analysis up to the log fold change table: quality control analysis with fastqc, UMI extraction with umi_tools extract, trimming with cutadapt, STAR alignment to the reference

genome of _S. pneumoniae_ D39, umi deduplication with umi_tools dedup and differential gene expression analysis with DESeq2. With the differential gene expression table from Lexogen and by

retrieving a STRING network setting the species to “_Streptococcus pneumoniae_ D39” in Cytoscape 3.8.0, we created a gene association network with genes ≥1.25 log2FC upregulated or ≤−1.25

log2FC downregulated. cDNA was synthesized using SuperScript IV Reverse transcriptase (Invitrogen), including RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen), according to the

manufacturer’s protocol. RT-qPCR was done according to the manufacturer’s protocol using TaqMan Fast Advanced Master Mix (Applied Biosystems) and Custom TaqMan Gene Expression Assays

(Applied Biosystems) with sequences in Supplementary Table 2. PROTEOMIC ANALYSIS Following overnight culture on agar plates, _S. pneumoniae_ strain D39 was grown in BHI + 5% FCS to OD600nm =

 0.5 and 100 µl of overnight culture was sub-cultured in 10 ml of fresh CDM until OD600nm = 0.2. The culture was split into two 5 ml cultures and 0.5 mg/ml peptide was added to one sample.

After 15 min incubation at 37 °C, both cultures were centrifuged at 4000×_g_ for 5 min at 4 °C. The pellet was washed in 1 ml PBS and transferred to an Eppendorf tube to centrifuge at

4000×_g_ for 5 min. The supernatant was removed and the pellet was snap frozen in liquid nitrogen. Mass spectrometry was performed at the Core Facility Proteomics and Mass Spectrometry

(University of Bern) as described as follows and elsewhere58. Bacterial pellets were lysed in 50 μl buffer containing 8 M urea and 100 mM Tris–HCl pH 8.0 and protease inhibitor cocktail

(cOmplete w/o EDTA, Roche). Protein concentration was determined with Qubit assay (ThermoFisher) and adjusted to 1 mg/ml with lysis buffer and an aliquot corresponding to 10 μg was

processed. Proteins were reduced by the addition of 1/10 volume of 0.1 M DTT and incubation for 30 min at 37 °C, followed by alkylation with a five-fold molar excess of iodoacetamide and

incubation for 30 min at 37 °C in the dark. Iodoacetamide was quenched by 4/10 volume of 0.1 M DTT and urea concentration lowered to 4 M by addition of 20 mM Tris/HCl pH 8.0 with 2 mM CaCl2.

Proteins were digested for 2 h at 37 °C with sequencing grade LysC (Promega) at a protein/protease ratio of 50:1, followed by urea dilution to 1.6 M and overnight digestion at ambient

temperature with 1:50 sequencing grade trypsin (Promega). Digestion was stopped with trifluoroacetic acid (TFA) to a final concentration of 1% (v/v), followed by dilution to 0.1 μg/μl of

protein. Proteome analysis was done by nano-liquid chromatography on a Dionex Ultimate 3000 (Thermo Fisher Scientific) through a CaptiveSpray source coupling to a timsTOF Pro mass

spectrometer (Bruker) with an end-plate offset of 500 V, a drying temperature of 200 °C, and with the capillary voltage fixed at 1.6 kV. A volume of 2 µl (200 ng) from the protein digest was

loaded onto a pre-column (PepMap 100 C18, 5 µm, 100 A, 300 µm diameter × 5 mm length, ThermoFisher) at a flow rate of 10 µl/min with 0.05% (v/v) TFA in water/acetonitrile 98:2. After

loading, peptides were eluted in back flush mode onto a home-made C18 CSH Waters column (1.7 µm, 130 Å, 75 µm × 20 cm) by applying a 90 min gradient of 5% acetonitrile to 40% in water/0.1%

formic acid, at a flow rate of 250 nl/min and data was acquired in data-dependent acquisition mode using the parallel acquisition serial fragmentation (PASEF) option. The mass range was set

between 100 and 1700 _m_/_z_, with 10 PASEF scans between 0.7 and 1.4 V s/cm2. The accumulation time was set to 2 ms and the ramp time to 100 ms, respectively. Fragmentation was triggered at

20,000 arbitrary units, and peptides (up to a charge of 5) were fragmented using collision-induced dissociation with a spread between 20 and 59 eV. DDA data was interpreted and processed

with MaxQuant (version 2.0.1.0) against the UniprotKB _S. pneumoniae_ D39 strain protein sequences (release February 2021) as described elsewhere58. GENERATION OF HUMAN AIRWAY EPITHELIAL

CELL (HAEC) CULTURES Human AEC cultures were cultured in 6.5 mm Transwells® with 0.4 µm pore polyester membrane insert in flat bottom 24-well plates (Corning, 3470) as described

previously21. We seeded 1 × 105 cells per insert. Experiments were performed once a pseudostratified layer of differentiated airway epithelial cells was formed, including cilia movement and

physiological mucus production. CYTOTOXICITY ASSAY IN HAEC CULTURES Cytotoxicity of 30 h peptide incubation on hAEC cultures was determined by measuring the release of lactate dehydrogenase

(LDH), which is a stable cytosolic enzyme released upon cell lysis, and visualizing the ultrastructure of the epithelium. To the apical side of hAEC cultures, 40 µl of TEER solution (NaCl

0.9%, CaCl2 1.25 mmol/l and HEPES 10 mmol/l dissolved in distilled water) were added as negative control, 40 µl of peptide resuspended in TEER solution to 0.25 mg/ml and 0.5 mg/ml and 40 µl

of 1x LDH+ control solution from the CytoTox® Non-Radioactive Cytotoxicity Assay (Promega) were added to the apical side. After 30 h of incubation, 160 µl TEER solution was added to the

apical side and the medium from the apical and basolateral side were collected and both samples of each culture were used for cytotoxicity determination. Release of LDH was measured to

determine the cytotoxicity of peptide V11A on hAEC cultures using the CytoTox® Non-Radioactive Cytotoxicity Assay (Promega) according to the manufacturer’s instructions. TOXICITY ASSAY IN

ZEBRAFISH LARVAE AB zebrafish eggs were kindly provided by Anna Gliwa, Institute of Anatomy (University of Bern), obtained by natural mating procedures. Zebrafish eggs were collected within

the first hours post fertilization (hpf) and eggs and larvae were maintained at 28.5 °C on a 14 h/10 h light/dark cycle in a dedicated incubator (IPP110plus, Memmert GmbH) and used for

experiments until no later than 5 days post fertilization (dpf). The fertilized eggs were kept for 1 day in E3 embryo medium (60× stock solution contains 34.8 g NaCl, 1.6 g KCl, 5.8 g CaCl2,

9.78 g MgCl2*6 H2O) supplemented with 0.3 mg/ml methylene blue. On the following day, the media was changed to E3 only. Unfertilized eggs and deformed embryos were discarded. Peptides were

resuspended in E3 to 0.5 mg/ml and 0.25 mg/ml. E3 was used as a negative control and 150 µg/ml LPS in E3 as a positive control for toxicity. Healthy larvae of similar developmental phases

were picked 3 dpf and 15 larvae were transferred to each well of a six-well plate. For peptide toxicity testing, 2 wells (i.e. 30 larvae) were used per concentration, and for positive and

negative control, one well each. E3 was replaced by 2 ml of E3 without or with LPS or peptide and larvae were incubated for 48 h at 28.5 °C with light/dark cycle as above. At 48 h a

DanioVision Observation Chamber (Nodus) dark/light protocol59 with 1 h adaptation time, followed by 20 min dark, 10 min light, and 20 min dark, was run to investigate whether peptide

treatment caused swimming behavioural changes with Ethovision XT 15 software. Larvae were euthanized after the experiment with 0.4% Tricaine. ADHERENCE ASSAY ON HAEC CULTURES Two days prior

to infection, the basolateral medium of hAEC cultures was changed to a fresh medium without antibiotics. Bacterial culture of _S. pneumoniae_ strain D39 was prepared as described for the

growth assay. On the day of infection, 500 µl of the overnight culture were sub-cultured in 5 ml of fresh BHI until OD600nm = 0.4, then centrifuged at 3000×_g_ for 7 min and the pellet was

resuspended in 5 ml TEER solution for one wash, before resuspending again in 5 ml fresh TEER solution and diluting the bacterial solution for inoculation of hAEC cultures to 1 × 106 cfu/ml.

AEC cultures were washed with TEER to remove mucus at the time of infection. Peptides were resuspended in bacterial inoculum solution to 0.5 mg/ml and hAEC cultures inoculated with 40 µl of

bacterial inoculum with or without peptides per insert (to give a final inoculum of 4 × 104 cfu/insert). Next, the hAEC cultures were incubated for 24 h at 37 °C in a humidified atmosphere

with 5% CO2 then three washes with 200 µl of TEER solution were performed apically. Human AEC cultures were detached by adding 100 µl TrypLETM Express Enzyme (1x), phenol red (Gibco) for 30 

min at 37 °C, 5% CO2 with resuspension after the first 10 min before continuing the incubation time. The trypsinized samples were collected into 100 µl of BHI with 5% FCS and directly

processed by making dilutions and plating them onto CSBA plates to determine cfu after overnight incubation at 37 °C, 5% CO2. RAT COLONIZATION MODEL Bacterial culture of _S. pneumoniae_

strain 1154.75 (serotype 23F) was prepared as described for the growth assay. On the day of inoculation, 500 µl of the overnight culture were sub-cultured in 5 ml of BHI until OD600nm = 0.4,

then centrifuged at 3000×_g_ for 7 min and the pellet washed in 0.85% NaCl solution before resuspending again, in 5 ml 0.85% NaCl solution for an inoculum of 5 × 107 cfu/ml. We performed

two experiments on different occasions: (1) two litters each of 12 (6 male, 6 female) nursing Wistar rats along with their dams (specific pathogen-free, SPF) were obtained from Charles River

(Germany), kept in individually ventilated cages with a 12 h light/dark cycle and constant temperature of 22 ± 2 °C, and provided with tap water and gamma-sterilized pellet diet ad libitum.

Once 14 days old, the rat pups were inoculated intranasally with the bacterial inoculum with or without 0.5 mg/ml peptide V11A by dripping 5 µl of inoculum into each nare, in alternate.

Separate litters of 12 pups were used for no peptide and peptide conditions. Then, 24 h after bacterial inoculation, 6 pups of each litter (3 male, 3 female) were sacrificed by an overdose

of pentobarbital (150 mg/kg, i.p.). Nasal wash was done by flushing through the trachea with 200 µl 0.85% NaCl solution and harvesting from the nostrils, and the nasopharynx was extracted.

For the remaining pups, 0.85% NaCl solution or 0.5 mg/ml V11A in 0.85% NaCl solution was administered again and 48 h after bacterial inoculation, sacrifice, nasal wash and nasopharynx

extraction were done as above. (2) The second experiment was done similarly to the first but with the following changes: one litter of 10 (5 male, 5 female) nursing Wistar rats along with

their dam were obtained and kept in a ventilated cage. Once 14 days old, the rat pups were inoculated intranasally with the bacterial inoculum with or without 0.5 mg/ml peptide V11A by

dripping 5 µl of inoculum into each nare, in alternate. All pups were kept in the same cage. Then, 24 h after bacterial inoculation, all pups were sacrificed and nasal wash and nasopharynx

extraction were performed as above. The extracted nasopharynxes were placed into 2 ml homogenization tubes with beads (2.8 mm diameter ceramic beads, BER0072, Labgene Scientific) containing

300 µl of PBS on ice and homogenized with 3 cycles of 30 s and 4500 rpm and 30 s break with Precellys Evolution homogenizer (Bertin Technologies). The nasal wash and nasopharynx samples were

directly processed by making dilutions and plating them onto CSBA plates with 3 µg/ml trimethoprim and 57 µg/ml sulfamethoxazole to determine cfu after overnight incubation of the plates at

37 °C, 5% CO2. STATISTICS All analyses were done in the R version 4.0.3 within R studio version 1.3.1093. Shapiro–Wilk normality tests were used to evaluate normality. If more than one

sample group was not normally distributed, the Wilcoxon rank sum test was done, otherwise _t_-test was used. As no cell of the expected frequencies in the contingency table was less than 5,

a Chi-squared test was performed to test for the effect of peptide V11A on number of rats colonized. In all analyses, a _p_-value of ≤0.05 was considered statistically significant. ETHICS

All research was performed in accordance with the relevant guidelines and regulations. In accordance with the Swiss Human Research Law, researchers at Bern University Hospital received

anonymized discarded leftovers of human cerebrospinal fluid samples collected in clinical routine. For the human primary airway epithelial cell model the anonymized post-mortem tissue

material was obtained through the Tissue Bank Bern (TBB) in accordance with ethical approval (KEK-BE 1571/2019). Studies with the rats were approved by the Animal Care and Experimentation

Committee of the Canton Bern, Switzerland, and follow the ethical principles and guidelines for experiments on animals as published by the Swiss Federal Veterinary Office, licence number

BE64/2022. REPORTING SUMMARY Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. DATA AVAILABILITY All data generated or

analysed during this study are included in this published article and supplementary files. The source data underlying the figures and Supplementary figures can be found in Supplementary Data

 1. The transcriptomic data for this study has been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB73628 and processed data (normalized counts and

differential gene expression analysis) are available in Supplementary Data 2. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE60

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Acids Res._ 50, D543–D552 (2021). Article  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We are grateful to the following people for providing pneumococcal strains:

Jeffrey Weiser (New York University, USA) for D39, Philippe Moreillon (Centre Hospitalier Universitaire Vaudois, Switzerland) for R6 and Ralf Reinert (National Reference Center for

Pneumococcus, Germany) for reference strains. We thank Carlo Casanova (Institute for Infectious Diseases, University of Bern) for kindly providing strains of _S. pneumoniae, H. influenzae,

S. aureus, M. catarrhalis_ and _K. pneumoniae_. We would like to thank the Next Generation Sequencing Platform, University of Bern, for performing the high-throughput sequencing experiments.

We are grateful to Sabina Berezowska and Irene Ramos-Centeno (Institute of Pathology, University of Bern) for providing the tissues for the hAEC culture establishment via the Tissue Bank

Bern. We thank Anna Gliwa (Institute for Anatomy, University of Bern) for providing zebrafish larvae eggs. We are grateful to Marianne Küffer, Adrian Wegmüller and Linus Rechsteiner

(Institute for Infectious Diseases, University of Bern), Sophie Braga-Lagache and Natasha Buchs (Proteomics and Mass Spectrometry Core Facility, University of Bern) and Zhian Salehian

(Norwegian University of Life Sciences) for their technical support. This work was funded by grant number 192067 from the Swiss National Science Foundation to L.J.H. We gratefully

acknowledge the Swiss National Science Foundation (R’Equip grant no. 316030-189737) and the University of Bern for financing the mass spectrometry equipment. AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Faculty of Medicine, Institute for Infectious Diseases, University of Bern, Bern, Switzerland Janine Lux, Hannah Portmann, Lucía Sánchez García, Maria Erhardt, Lalaina

Holivololona, Laura Laloli, Manon F. Licheri, Ronald Dijkman, Denis Grandgirard, Stephen L. Leib & Lucy J. Hathaway * Graduate School for Cellular and Biomedical Sciences, University of

Bern, Bern, Switzerland Janine Lux * Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland Clement Gallay & Jan-Willem Veening * Department of Neurology,

Bern University Hospital and University of Bern, Bern, Switzerland Robert Hoepner * MRC Centre for Global Infectious Disease Analysis, Sir Michael Uren Hub, White City Campus, Imperial

College London, London, UK Nicholas J. Croucher * Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, 1430, Ås, Norway Daniel Straume * Proteomics

and Mass Spectrometry Core Facility, Department for BioMedical Research (DBMR), University of Bern, Bern, Switzerland Manfred Heller Authors * Janine Lux View author publications You can

also search for this author inPubMed Google Scholar * Hannah Portmann View author publications You can also search for this author inPubMed Google Scholar * Lucía Sánchez García View author

publications You can also search for this author inPubMed Google Scholar * Maria Erhardt View author publications You can also search for this author inPubMed Google Scholar * Lalaina

Holivololona View author publications You can also search for this author inPubMed Google Scholar * Laura Laloli View author publications You can also search for this author inPubMed Google

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inPubMed Google Scholar * Robert Hoepner View author publications You can also search for this author inPubMed Google Scholar * Nicholas J. Croucher View author publications You can also

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Heller View author publications You can also search for this author inPubMed Google Scholar * Denis Grandgirard View author publications You can also search for this author inPubMed Google

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inPubMed Google Scholar CONTRIBUTIONS L.J.H. conceived the study, J.L. wrote the first draft of the manuscript. J.L., L.J.H., D.G. and S.L.L. participated in the design of the study. J.L.,

H.P. and L.S.G. did bacterial growth assays. D.S. provided D39 parental strain and D39∆_amiC_ and R.H. provided hCSF. J.L. did epifluorescence microscopy after preliminary experiments and

establishment of a protocol with C.G. and J.W.V. J.L. analysed bacterial cell shape, chain length, transformation rate, RNA-Seq and proteomics. N.J.C. provided support for the analysis of

RNA-Seq data in R. M. He. analysed mass spectrometry data. J.L., L.L., M.F.L. and R.D. performed or advised on hAEC culture assays. L.H. did cytotoxicity assay with zebrafish larvae. J.L.,

M.E. and D.G. performed experiments with infant rats. All authors gave final approval for publication. CORRESPONDING AUTHOR Correspondence to Lucy J. Hathaway. ETHICS DECLARATIONS COMPETING

INTERESTS The authors declare the following competing interests: L.J.H. and J.L. are inventors on a patent application pertaining to this work. R.H. received speaker/advisor honorary from

Merck, Novartis, Roche, Biogen, Alexion, Sanofi, Janssen, Bristol-Myers Squibb, Teva/Mepha and Almirall. He received research support within the last 5 years from Roche, Merck, Sanofi,

Biogen, Chiesi, and Bristol-Myers Squibb. He also received research grants from the Swiss MS Society and the SITEM Insel Support Fund and is a member of the Advisory Board of the Swiss and

International MS Society. He also serves as deputy editor-in-chief for the Journal of Central Nervous System Disease. All conflicts are not related to this work. All other authors declare no

competing interests. PEER REVIEW PEER REVIEW INFORMATION _Communications Biology_ thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling

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THIS ARTICLE CITE THIS ARTICLE Lux, J., Portmann, H., Sánchez García, L. _et al._ _Klebsiella pneumoniae_ peptide hijacks a _Streptococcus pneumoniae_ permease to subvert pneumococcal growth

and colonization. _Commun Biol_ 7, 425 (2024). https://doi.org/10.1038/s42003-024-06113-9 Download citation * Received: 22 November 2023 * Accepted: 26 March 2024 * Published: 08 April 2024

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